Hybridization kinetics of out-of-equilibrium mixtures of short RNA oligonucleotides

Hybridization and strand displacement kinetics determine the evolution of the base paired configurations of mixtures of oligonucleotides over time. Although much attention has been focused on the thermodynamics of DNA and RNA base pairing in the scientific literature, much less work has been done on the time dependence of interactions involving multiple strands, especially in RNA. Here we provide a study of oligoribonucleotide interaction kinetics and show that it is possible to calculate the association, dissociation and strand displacement rates displayed by short oligonucleotides (5nt–12nt) that exhibit no expected secondary structure as simple functions of oligonucleotide length, CG content, ΔG of hybridization and ΔG of toehold binding. We then show that the resultant calculated kinetic parameters are consistent with the experimentally observed time dependent changes in concentrations of the different species present in mixtures of multiple competing RNA strands. We show that by changing the mixture composition, it is possible to create and tune kinetic traps that extend by orders of magnitude the typical sub-second hybridization timescale of two complementary oligonucleotides. We suggest that the slow equilibration of complex oligonucleotide mixtures may have facilitated the nonenzymatic replication of RNA during the origin of life.     

Isolation and in vitro translation of polysomes from mature rye leaves

Cytoplasmic polysomes have been prepared from mature leaves of winter rye (Secale cereale L. cv Puma). This is the first time a method has been developed for isolation of highly polymerized polysomes from mature leaves. The degree of intactness of isolated plant polysomes has been determined by two independent but complementary methods: size class distribution by sucrose gradient centrifugation and in vitro translation. The polymerization of isolated polysomes was estimated by the ratio of the proportion of large polysomes to the proportion of small polysomes obtained from the profiles. Our results show that the composition of the optimal polysome isolation buffer for mature rye leaves is different from that reported for young tobacco and pea leaves. Polysomes were translated in vitro with the S-105 wheat germ fraction. The degree of polysome polymerization has a significant effect on their in vitro translation since both the incorporation of amino acid and the presence of high molecular weight polypeptides are proportional to the large polysomes/small polysomes ratio. This study emphasizes the need to evaluate isolation conditions carefully before proceeding with polysome studies in any particular tissue or in tissues under different physiological status.     

The Role of Ribulose-1,5-Bisphosphate Regeneration in the Induction Requirement of Photosynthetic CO(2) Exchange under Transient Light Conditions

The temporally variable light environment of natural plant canopies presents distinct limitations to carbon assimilation, partially as a result of the photosynthetic induction requirement that develops when leaves are shaded. This study was undertaken with soybean (Glycine max L.) leaves to further identify factors contributing to the activation state of the fast component of induction during low photosynthetic photon flux density (PPFD) periods. Determination of pool sizes of carbon reduction cycle intermediates at low light and upon return to saturating light indicated that different limitations to photosynthetic activity arise over the time course of a 10-minute low PPFD period. Photosynthetic activity upon reillumination was limited by the regeneration of ribulose 1,5-P₂. There was an increase in the levels of fructose 1,6-P₂, sedoheptulose 1,7-P₂, triose-P, ribose 5-P, and ribulose 5-P pools, indicating inactivation of stromal enzymes, most notably fructose 1,6-bisphosphatase, sedoheptulose 1,7-bisphosphatase, and ribulose 5-P kinase. The fast-induction component was the most important factor limiting assimilation during rapid, brief light transients, during which the decay of the slow component was minimal. This may be particularly significant for upper leaves in soybean canopies that generally experience very rapid light transients.     

Large-scale rotational motions of proteins detected by electron paramagnetic resonance and fluorescence.

Direct spectroscopic measurements of rotational motions of proteins and large protein segments are crucial to understanding the molecular dynamics of protein function. Fluorescent probes and spin labels attached to proteins have proved to be powerful tools in the study of large-scale protein motions. Fluorescence depolarization and conventional electron paramagnetic resonance (EPR) are applicable to the study of rotational motions in the nanosecond-to-microsecond time range, and have been used to demonstrate segmental flexibility in an antibody and in myosin. Very slow rotational motions, occurring in the microsecond-to-millisecond time range, are particularly important in supramolecular assemblies, where protein motions are restricted by association with other molecules. Saturation transfer spectroscopy (ST-EPR), a recently developed electron paramagnetic resonance (EPR) technique that permits the detection of rotational correlation times as long as 1 ms, has been used to detect large-scale rotational motions of spin-labeled proteins in muscle filaments and in membranes, providing valuable insights into energy transduction mechanisms in these assemblies.     

The rate of functional sperm transport into the oviducts of mated cows

The technique of ligation and transection of the oviducts from the uterus, together with subsequent examination of the eggs, has been used to establish how soon after mating a population of spermatozoa competent to fertilise enters the oviducts. Animals were mated within 8 h of the onset of oestrus, and the uterotubal junction ligated under local anaesthesia 6, 8 or 12 h later.Nineteen eggs were recovered from 29 animals. Fertilisation was not found as a sequel to ligation 6 h after mating, whereas 3 of 6 and 5 of 6 eggs, respectively, were fertilised when the operation was performed 8 or 12 h after mating. The mean number of spermatozoa associated with the zona pellucida increased over the interval examined from 0.2 to 4.5 (range 0–11). These results suggest that only a slow progression and displacement of viable spermatozoa occurs in the female tract after mating early in oestrus. They also infer that the functional sperm reservoir — the one drawn on at the time of ovulation — is in the oviductal isthmus rather than in the cervix, a consideration that may have a bearing on procedures of artificial insemination.     

Analysis of Sub-τc and Supra-τc Motions in Protein Gβ1 Using Molecular Dynamics Simulations

The functions of proteins depend on the dynamical behavior of their native states on a wide range of timescales. To investigate these dynamics in the case of the small protein Gβ1, we analyzed molecular dynamics simulations with the model-free approach of nuclear magnetic relaxation. We found amplitudes of fast timescale motions (sub-τ_c, where τ_c is the rotational correlation time) consistent with S² obtained from spin relaxation measurements as well as amplitudes of slow timescale motions (supra-τ_c) in quantitative agreement with S² order parameters derived from residual dipolar coupling measurements. The slow timescale motions are associated with the large variations of the ³J couplings that follow transitions between different conformational substates. These results provide further characterization of the large structural fluctuations in the native states of proteins that occur on timescales longer than the rotational correlation time.     

Protein dynamics of a β-sheet protein

Rhodnius prolixus Nitrophorin 4 (abbreviated NP4) is an almost pure β-sheet heme protein. Its dynamics is investigated by X-ray structure determination at eight different temperatures from 122 to 304 K and by means of Mössbauer spectroscopy. A comparison of this β-sheet protein with the pure α-helical protein myoglobin (abbreviated Mbmet) is performed. The mean square displacement derived from the Mössbauer spectra increases linearly with temperature below a characteristic temperature T _c. It is about 10 K larger than that of myoglobin. Above T _c the mean square displacements increase dramatically. The Mössbauer spectra are analyzed by a two state model. The increased mean square displacements are caused by very slow motions occurring on a time scale faster than 140 ns. With respect to these motions NP4 shows the same protein specific modes as Mbmet. There is, however, a difference in the fast vibration regime. The B values found in the X-ray structures vary linearly over the entire temperature range. The mean square displacements in NP4 increase with slopes which are 60% larger than those observed for Mbmet. This indicates that nitrophorin has a larger structural distribution which makes it more flexible than myoglobin.     

Anisotropic rotational diffusion in model-free analysis for a ternary DHFR complex

Model-free analysis has been extensively used to extract information on motions in proteins over a wide range of timescales from NMR relaxation data. We present a detailed analysis of the effects of rotational anisotropy on the model-free analysis of a ternary complex for dihydrofolate reductase (DHFR). Our findings show that the small degree of anisotropy exhibited by DHFR (D_||/D=1.18) introduces erroneous motional models, mostly exchange terms, to over 50% of the NH spins analyzed when isotropic tumbling is assumed. Moreover, there is a systematic change in S², as large as 0.08 for some residues. The significant effects of anisotropic rotational diffusion on model-free motional parameters are in marked contrast to previous studies and are accentuated by lowering of the effective correlation time using isotropic tumbling methods. This is caused by the preponderance of NH vectors aligned perpendicular to the principal diffusion tensor axis and is readily detected because of the high quality of the relaxation data. A novel procedure, COPED (COmparison of Predicted and Experimental Diffusion tensors) is presented for distinguishing genuine motions from the effects of anisotropy by comparing experimental relaxation data and data predicted from hydrodynamic analyses. The procedure shows excellent agreement with the slow motions detected from the axially symmetric model-free analysis and represents an independent procedure for determining rotational diffusion and slow motions that can confirm or refute established procedures that rely on relaxation data. Our findings show that neglect of even small degrees of rotational diffusion anisotropy can introduce significant errors in model-free analysis when the data is of high quality. These errors can hinder our understanding of the role of internal motions in protein function.     

Sampling Plant Diversity and Rarity at Landscape Scales: Importance of Sampling Time in Species Detectability

Documenting and estimating species richness at regional or landscape scales has been a major emphasis for conservation efforts, as well as for the development and testing of evolutionary and ecological theory. Rarely, however, are sampling efforts assessed on how they affect detection and estimates of species richness and rarity. In this study, vascular plant richness was sampled in 356 quarter hectare time-unlimited survey plots in the boreal region of northeast Alberta. These surveys consisted of 15,856 observations of 499 vascular plant species (97 considered to be regionally rare) collected by 12 observers over a 2 year period. Average survey time for each quarter-hectare plot was 82 minutes, ranging from 20 to 194 minutes, with a positive relationship between total survey time and total plant richness. When survey time was limited to a 20-minute search, as in other Alberta biodiversity methods, 61 species were missed. Extending the survey time to 60 minutes, reduced the number of missed species to 20, while a 90-minute cut-off time resulted in the loss of 8 species. When surveys were separated by habitat type, 60 minutes of search effort sampled nearly 90% of total observed richness for all habitats. Relative to rare species, time-unlimited surveys had ∼65% higher rare plant detections post-20 minutes than during the first 20 minutes of the survey. Although exhaustive sampling was attempted, observer bias was noted among observers when a subsample of plots was re-surveyed by different observers. Our findings suggest that sampling time, combined with sample size and observer effects, should be considered in landscape-scale plant biodiversity surveys.     

Slow motions in oriented phospholipid bilayers and effects of cholesterol or gramicidin. A 19F-NMR T1 rho study.

In an extension of our earlier work (Peng, Z.-y., V. Simplaceanu, I. J. Lowe, and C. Ho. 1988. Biophys. J. 54:81-95), the rotating-frame nuclear spin-lattice relaxation (T1 rho) technique has been used to investigate the slow molecular motions (10(-4) - 10(-6) s) in lipid bilayers prepared from protonated or perdeuterated 19F-labeled phospholipids in the absence and presence of cholesterol or gramicidin as membrane-interacting molecules. Complications caused by the 19F-1H cross-polarization observed previously can be removed by the substitution of 2H for 1H in the acyl chains. Only a weak dependence of the T-1(1 rho) on the locking field strength is found for a phospholipid molecule with perdeuterated acyl chains, indicating that there are no slow motions with a single, well-defined correlation time between 5 x 10(-6) and 4 x 10(-5) s. However, the orientation dependences of the T-1(1 rho) can be well fitted by motional models with either one slow motion having an unspecified geometry or with a superposition of two specific types of slow motions. Cholesterol and gramicidin show distinct effects in altering either the geometry or the weighting of slow motions in phospholipid bilayers, as reflected by changes in the orientation dependence. These two additives also exhibit quite different label-position specificities. A qualitative understanding of the induced effects of cholesterol and gramicidin on the dynamics of phospholipid bilayers will be discussed.     

Continuous dental eruption identifies Sts 5 as the developmentally oldest fossil hominin and informs the taxonomy of Australopithecus africanus

The relatively small Australopithecus africanus specimen Sts 5 has figured prominently in taxonomic debates, and the determination of this specimen as a young male or an elderly female has the potential to offer a great deal of resolution on this question. Sts 5 has been argued to be either a small, immature male or a mature female based on a variety of characters.     

Studies on the behavior of the senescence signal in anoka soybeans

Soybean, a monocarpic plant, has been found to undergo rapid senescence as its fruits mature. In soybeans (Glycine max[L.] Merrill) cv. Anoka, foliar senescence begins during the period of most rapid pod-fill (seed growth), and it can be eliminated by surgical removal of the seeds at an early stage of their growth. Experiments in which fruits are removed from some regions of the plant but allowed to remain on other regions have established that the transmission of the senescence signal is limited; it affects mainly those leaves nearest to the nodes bearing the fruits. The implications of this localized signal movement are discussed.     

Physiological responses at short distances from a parametric speaker

In recent years, parametric speakers have been used in various circumstances. In our previous studies, we verified that the physiological burden of the sound of parametric speaker set at 2.6 m from the subjects was lower than that of the general speaker. However, nothing has yet been demonstrated about the effects of the sound of a parametric speaker at the shorter distance between parametric speakers the human body. Therefore, we studied this effect on physiological functions and task performance. Nine male subjects participated in this study. They completed three consecutive sessions: a 20-minute quiet period as a baseline, a 30-minute mental task period with general speakers or parametric speakers, and a 20-minute recovery period. We measured electrocardiogram (ECG) photoplethysmogram (PTG), electroencephalogram (EEG), systolic and diastolic blood pressure. Four experiments, one with a speaker condition (general speaker and parametric speaker), the other with a distance condition (0.3 m and 1.0 m), were conducted respectively at the same time of day on separate days. To examine the effects of the speaker and distance, three-way repeated measures ANOVA (speaker factor x distance factor x time factor) were conducted. In conclusion, we found that the physiological responses were not significantly different between the speaker condition and the distance condition. Meanwhile, it was shown that the physiological burdens increased with progress in time independently of speaker condition and distance condition. In summary, the effects of the parametric speaker at the 2.6 m distance were not obtained at the distance of 1 m or less.     

Psychophysiological responsivity on a laboratory stress task: methodological implications for a stress-muscle hyperactivity pain model

A stress-muscle hyperactivity-pain (SMP) model has been proposed to explain the etiology of certain musculoskeletal pain disorders. According to this model, subjects should show physiological arousal during periods of stress relative to periods of rest. In a test of this prediction, 31 subjects performed a reaction time task that has been used in previous laboratory studies. Multiple psychophysiological variables were monitored during initial and final 10-minute baselines, during performance on nine 2-minute reaction time tasks, and during 36-second rest intervals following each of the 2-minute tasks, Results showed small but statistically significant differences generally supporting the SMP model when masseter EMG was averaged over time periods of 12 seconds to 2 minutes, but not when masseter EMG was averaged over 10- to 18-minute blocks. These results demonstrated the importance of carefully selecting time intervals for analysis. Additional analyses that compared TMD with symptom-free subjects revealed small differences in EMG that supported the SMP model. Analyses of EMG over shorter time intervals also showed, however, that masseter EMG increased during the 36-second rest interval following performance on a 2-minute stress task; this result suggested that a modification of the SMP model may be necessary.     

Model for Short Term Movements in Stapelia variegata Linn

Recently it was demonstrated that holographic interferometry could be used to quantify minute displacements of a mature Stapelia variegata Linn. The previously unobtainable measurements revealed that the short term response of the stapelia was regular enough so that the dynamics could be evaluated through the use of a highly damped mass, spring, dashpot model. Such a model is derived explicitly and the relationship between movements of the entire plant and the dynamics of individual cells or groups of cells is shown.     

Composition of gum exudates from Anacardium occidentale

The composition and solution properties of Indian and Papuan specimens of the gum from Anacardium occidentale have been studied and found to be closely similar. Contrary to earlier reports by Indian workers, this gum does not contain galacturonic acid. It does, however, contain glucose; this appears to be the first report of the presence of this sugar in a plant gum exudate. A freeze-dried sample of the gum was examined at intervals over a period of 2 months; its weight-average MW increased by a factor of three in that time, and molecular-sieve chromatography showed that self-association occurred with the formation of a small proportion of a very high MW component.     

Differential behavior of liposome-introduced specific RNAs in living Drosophila cells

We have developed a protocol for efficiently introducing macromolecules into Drosophila tissue culture cells using liposomes. By carefully adjusting the fusion parameters, conditions have been established to routinely encapsulate 15–30% of the starting material into liposomes and to introduce 20–30% of the liposome-encapsulated material into the cells during a 30-minute fusion period. Essentially, all of the cells receive material from the liposomes and 10⁹ cells can be fused at once. The fusion does not have any measureable effect on cell viability as assayed by trypan blue exclusion, growth rate, and cell morphology. We have utilized this technique to introduce radioactive RNAs into nonradioactive cells, thus enabling the behavior of the introduced RNAs to be followed unambiguously. Liposome-introduced small nuclear RNAs (snRNAs) are stable in the cell for at least 25 hours (approximately two cell generations), with 80% of the radioactivity remaining trichloroacetic acid (TCA) precipitable and the gel electrophoresis pattern remaining essentially unchanged. This is in contrast to liposome-introduced cytoplasmic RNAs, which are only 20% TCA precipitable after the first hour. In the cell, the introduced snRNAs attain a 10–35-fold higher concentration in the nucleus than the cytoplasm. Nuclear accumulation is not seen with Drosophila tRNA or 5s RNA, both of which attain the same nuclear as cytoplasmic RNA concentration.     

An analysis of species response curves and of competition from field data: Some results from heath vegetation

The variation in species composition of plant communities has been described by means of the Gaussian distribution (relations between plant populations and environmental variables) and by linear equations (mutual interference between mixed populations). An important presupposition is the existence of a theoretical population, covering 100% of the soil surface. A numerical approximation of the parameters can be obtained by means of an iterative procedure. This technique has been applied to heath vegetation. A small sample has been used to demonstrate the algorithm. The cover ofCalluna vulgaris, Erica tetralix andMolinia caerulea could be explained to a large extent from phosphate and ground water table. A comparison of the results with those of experiments by other authors showed a reasonable similarity. The linear coefficients of interference as means to describe relations between mixed plant populations in a local area have been evaluated by means of dynamic simulation.     

Effects of Viscosity and Constraints on the Dispersion and Dissipation of Waves in Large Blood Vessels: I. Theoretical Analysis

The propagation of sounds and pulse waves within the cardiovascular system is subject to strong dissipative mechanisms. To investigate the effects of blood viscosity on dissipation as well as dispersion of small waves in arteries and veins, a parametric study has been carried out. A linearized analysis of axisymmetric waves in a cylindrical membrane that contains a viscous fluid indicates that there are two families of waves: a family of slow waves and one of fast waves. The faster waves are shown to be more sensitive to variations in the elastic properties of the medium surrounding the blood vessels and at high values of the frequency parameter α defined by α = √ρωR²₀/μ the blood viscosity attenuates them more strongly over a length than the slow waves. At low values of α, the effects of viscosity on attenuation are reversed; that is, the family of slow waves is much more attenuated than the family of fast waves. For the slow waves the radial displacement component generally exceeds the axial component except at very low frequencies. Conversely the axial displacements are much larger than the radial displacement for the faster waves. The presence of external constraints, however, can modify these results. In the case of the slow waves the phase angle between pressure and radial wall displacement is virtually negligible in the presence of mild external constraints, while the phase angles between pressure and fluid mass flow are at most 45°. The corresponding phase angles for the fast waves exhibit much larger variations with changes in the elastic properties of the surrounding medium.