C-reactive Protein Exists in an NaCl Concentration-dependent Pentamer-Decamer Equilibrium in Physiological Buffer

C-reactive protein (CRP) is an acute phase protein of the pentraxin family that binds ligands in a Ca²⁺-dependent manner, and activates complement. Knowledge of its oligomeric state in solution and at surfaces is essential for functional studies. Analytical ultracentrifugation showed that CRP in 2 mm Ca²⁺ exhibits a rapid pentamer-decamer equilibrium. The proportion of decamer decreased with an increase in NaCl concentration. The sedimentation coefficients s_20,w⁰ of pentameric and decameric CRP were 6.4 S and in excess of 7.6 S, respectively. In the absence of Ca²⁺, CRP partially dissociates into its protomers and the NaCl concentration dependence of the pentamer-decamer equilibrium is much reduced. By x-ray scattering, the radius of gyration R_G values ranged from 3.7 nm for the pentamer to above 4.0 nm for the decamer. An averaged K_D value of 21 μm in solution (140 mm NaCl, 2 mm Ca²⁺) was determined by x-ray scattering and modeling based on crystal structures for the pentamer and decamer. Surface plasmon resonance showed that CRP self-associates on a surface with immobilized CRP with a similar K_D value of 23 μm (140 mm NaCl, 2 mm Ca²⁺), whereas CRP aggregates in low salt. It is concluded that CRP is reproducibly observed in a pentamer-decamer equilibrium in physiologically relevant concentrations both in solution and on surfaces. Both 2 mm Ca²⁺ and 140 mm NaCl are essential for the integrity of CRP in functional studies and understanding the role of CRP in the acute phase response.     

Use of lipophilic cations to measure the membrane potential of oat leaf protoplasts

Uptake of the lipophilic cation triphenylmethylphosphonium into mesophyll protoplasts of oat (Avena sativa L. cv. “Garry”) approaches equilibrium at 3 to 4 hours. The resulting external and internal concentrations are then used with the Nernst equation to obtain a membrane potential of −62 millivolts, inside negative. Potentials calculated in this manner are depolarized by adding 2 mm sodium azide and 50 μm carbonyl cyanide m-chlorophenylhydrazone as well as by increasing the external proton and potassium concentrations. The depolarizations are qualitatively similar to those seen when oat mesoyphll cells are measured in situ with microelectrodes. It is concluded that due to the lack of turgor and fragility of protoplasts, estimations of their membrane potential may be made more reliably, under some conditions, with lipophilic cations than with microelectrodes.     

Chloride accumulation by mung bean root tips: a low affinity active transport system at the plasmalemma

Net uptake of Cl⁻ into root tips of mung bean (Phaseolus aureus) increases steadily with increasing external concentrations from 1 to 60 mm. Membrane potentials were measured to determine the equilibrium concentration of Cl⁻ in the tissue which could be due to diffusion. This concentration was readily exceeded in both the relatively nonvacuolate tips (0 to 1 mm) and the vacuolate, mature upper sectons (1 to 11 mm) of the roots. The activity coefficient of both cytoplasmic and vacuolar Cl⁻, measured with Cl⁻ sensitive microelectrodes, was approximately the same as that of a pure KCl solution of the same concentration. It is concluded that the “second mechanism” of ion uptake involves a large increase in the rate of active transport at the plasmalemma as the external concentration is increased above 1 mm.     

Complexity in matrix population models: Polyvariant ontogeny and reproductive uncertainty

Linear matrix models of stage-structured population dynamics are widely used in plant and animal demography as a tool to evaluate the growth potential of a population in a given environment. The potential is identified with λ₁, the dominant eigenvalue of the projection matrix, which is compiled of stage-specific transition and fertility rates. Advanced botanical studies reveal polyvariant ontogeny in perennial plants, i.e., multiple different versions of individual development within a local population of a single species. This phenomenon complicates any standard, successive-stage, life cycle graph to a digraph defined on a 2D lattice in the age and stage dimensions, the pattern of projection matrix becoming more complex too. In a kind of experimental design, the transition rates can be calculated directly from the data for two successive time moments, but the age-stage-specific rates of reproduction still remain uncertain, adding more complexity to the calibration problem. Simple additional assumptions could technically eliminate the uncertainty, but they contravene the biology of a species in which polyvariant ontogeny is considered to be the major mechanism of adaptation. Given the data and expert constraints, the calibration can be reduced instead to a nonlinear maximization problem, yet with linear constraints. I prove that it has a unique solution to be attained at a vertex of the constraint polyhedral. To facilitate searching for the solution in practice, I use the net reproductive rate R₀, a well-known indicator for the principal property of λ₁ to be greater or less than 1. The method is exemplified with the calibration of a projection matrix in an age-stage-structured model (published elsewhere) for Calamagrostis canescens, a perennial herbaceous species with a complex (multivariant) life cycle that features unlimited growth when colonizing open areas.     

Unidirectional fluxes in saturated single-file pores of biological and artificial membranes. II. Asymptotic behavior at high degrees of saturation

A rate-theory analysis of isotope fluxes in filled single-file pores is given. It is assumed that the relation between the activities in solution and the fluxes can be described by the asymptotic laws for high activities. Only pore states containing no more than two vacancies need be taken into account. The flux-ratio exponent n, which is obtained by representing the ratio of unidirectional fluxes as a power of the electrochemical activity ratio, is found to provide information about m, the number of sites per pore. The general relation between n and m, for m ≥ 4, is m ? 3 ≤ n ≤ m ?1. For m = 2 or 3 the minimum value is 1. The theoretical results are discussed with respect to the internal structure of gramicidin pores in artificial bilayer membranes.     

Steady-State Analysis and Scheduling of Cyclic Job Shops with Overtaking

A cyclic shop is a production system that repeatedly produces identical sets of parts of multiple types, called minimal part sets (MPSs), in the same loading and processing sequence. A different part type may have a different machine visit sequence. We consider a version of cyclic job shop where some operations of an MPS instance are processed prior to some operations of the previous MPS instances. We call such a shop an overtaking cyclic job shop (OCJS). The overtaking degree can be specified by how many MPS instances the operations of an MPS instance can overtake. More overtaking results in more work-in-progress, but reduces the cycle time, in general. We prove that for a given processing sequence of the operations at each machine, under some conditions, an OCJS has a stable earliest starting schedule such that each operation starts as soon as its preceding operations are completed, the schedule repeats an identical timing pattern for each MPS instance, and the cycle time is kept to be minimal. To do these, we propose a specialized approach to analyzing steady states for an event graph model of an OCJS that has a cyclic structure, which can keep the MPS-based scheduling concept. Based on the steady-state results, we develop a mixed integer programming model for finding a processing sequence of the operations at each machine and the overtaking degrees, if necessary, that minimize the cycle time.     

Reducing Competitive Suppression of a Rare Annual Forb by Restoring Native California Perennial Grasslands

Populations of the rare annual forb Amsinckia grandiflora may be declining because of competitive suppression by exotic annual grasses, and may perform better in a matrix of native perennial bunchgrasses. We conducted a field competition experiment in which Amsinckia seedlings were transplanted into forty 0.64‐m² experimental plots of exotic annual grassland or restored perennial grassland. The perennial grassland plots were restored using mature 3 cm‐diameter plants of the native perennial bunchgrass Poa secunda planted in three densities. The exotic annual grassland plots were established in four densities through manual removal of existing plants. Both grass types reduced soil water potential with increasing biomass, but this reduction was not significantly different between grass types. Both grass types significantly reduced the production of Amsinckia inflorescences. At low and intermediate densities (dry biomass per unit area of 20–80 g/m²), the exotic annual grasses reduced Amsinckia inflorescence number to a greater extent than did Poa, although at high densities (>90 g/m²) both grass types reduced the number of Amsinckia inflorescences to the same extent. The response of Amsinckia inflorescence number to Poa biomass was linear, whereas the same response to the annual grass biomass is logarithmic, and appeared to be related to graminoid cover. This may be because of the different growth forms exhibited by the two grass types. Results of this research suggest that restored native perennial grasslands at intermediate densities have a high habitat value for the potential establishment of the native annual A. grandiflora.     

A note on productivity gains in flexible robotic cells

Flexible robotic cells combine the capabilities of robotic flow shops with those of flexible manufacturing systems. In an m-machine flexible cell, each part visits each machine in the same order. However, the m operations can be performed in any order, and each machine can be configured to perform any operation. We derive the maximum percentage increase in throughput that can be achieved by changing the assignment of operations to machines and then keeping that assignment constant throughout a lot's processing. We find that no increase can be gained in two-machine cells, and that the gain in three- and four-machine cells each is at most 14 \(\frac{2}{7}\)%.     

A new fast algorithm for solving the minimum spanning tree problem based on DNA molecules computation

The minimum spanning tree (MST) problem is to find minimum edge connected subsets containing all the vertex of a given undirected graph. It is a vitally important NP-complete problem in graph theory and applied mathematics, having numerous real life applications. Moreover in previous studies, DNA molecular operations usually were used to solve NP-complete head-to-tail path search problems, rarely for NP-hard problems with multi-lateral path solutions result, such as the minimum spanning tree problem. In this paper, we present a new fast DNA algorithm for solving the MST problem using DNA molecular operations. For an undirected graph with n vertex and m edges, we reasonably design flexible length DNA strands representing the vertex and edges, take appropriate steps and get the solutions of the MST problem in proper length range and O(3m + n) time complexity. We extend the application of DNA molecular operations and simultaneity simplify the complexity of the computation. Results of computer simulative experiments show that the proposed method updates some of the best known values with very short time and that the proposed method provides a better performance with solution accuracy over existing algorithms.     

A rapid sampling method for measuring efflux from erythrocytes.

A method is described for obtaining kinetic data using a water-jacketed Hamilton gas-tight syringe as a reaction vessel and delivering aliquots of the reaction mixture to a quench solution at intervals as small as 2 sec apart by means of a repeating dispenser attachment. This apparatus has been used to measure the rate of [¹⁴C]uridine equilibrium exchange efflux from human erythrocytes at 15°C and at uridine concentrations near the K_m for the transport process. It should be useful for kinetic studies of any reaction having a half time of the order of 4 sec or more, provided a method of rapidly quenching the reaction is available.     

Demiarcs,creaons and genons

Useful insights into the representation of natural systems can be gained by decomposing directed graphs (digraphs) into elementary components. Arcs of digraphs can be split into male demiarcs (outarcs) which leave vertices and female demiarcs (inarcs) which enter demiarcs. Likewise, a vertex can be split into an input perceiving side called the creaon and an output generating side called the genon. Digraphs can be regarded as being hierarchically organized because each vertex in a level-1 digraph can be expanded into a level-2 digraph. In general, each vertex of a level-i digraph can be expanded into a level-(i+1) digraph. Arcs of a level-i digraph can be regarded as bundles of level-(i + 1) arcs which are split at the vertex boundary. These elementary graphical components are shown to be useful for depicting input-output systems such as organisms, ecosystems and societies.     

Isolation and Characterization of Inner Membrane-Associated and Matrix NAD-Specific Isocitrate Dehydrogenase in Potato Mitochondria

The isotherm for isocitrate oxidation by potato (Solanum tuberosum L. var. Russet Burbank) mitochondria in the presence of exogenous NAD is characterized by a hyperbolic phase at isocitrate concentrations below 3 millimolar, and a sigmoid, or positively cooperative phase from approximately 3 to 30 millimolar. The two forms of isocitrate dehydrogenase were separated and characterized following the sonication of mitochondria in 15% glycerol in the absence of buffer, followed by fractionation in a density step gradient to yield inner membrane and matrix components. The membrane-associated isocitrate dehydrogenase was found to have a Hill, or cooperativity, number of 1, while the Hill number of the matrix enzyme was 2.5. Upon digitonin extraction the cooperativity number of the membrane enzyme rose to 3.5. The isocitrate K_m for the membrane enzyme was calculated to be approximately 5.9 × 10⁻⁴ molar, while the S_0.5 for the matrix was 6.9 × 10⁻⁴ molar. The NAD K_m for both enzymes was 150 micromolar. Whereas the membrane enzyme proved indifferent to adenine nucleotides, the matrix enzyme was arguably inhibited by AMP and ADP, and inhibited some 25% by 5 millimolar ATP. Both enzymes were negatively responsive to the mole fraction of NADH, the membrane enzyme being 50% inhibited at a mole fraction of 0.26, and the matrix enzyme by a mole fraction of 0.32. The suggestion is offered that the enzymes in question constitute two forms of a single enzyme, one peripherally associated with the inner membrane, and one soluble in the matrix. It is proposed that a degree of regulation may be achieved by the apportionment of the enzyme between the bound and free forms.     

Optimization of mass spectral features in MALDI-TOF MS profiling of Acinetobacter species

The influence of the matrix solution, sample form and deposition technique on the quality MALDI-TOF mass spectra was examined and assessed with the aim to improve MALDI-TOF MS performance for the identification of microorganisms and to enable automatic spectra acquisition. It was observed that the use of matrix compounds ferulic and sinapinic acid may result in improved mass spectral features, in terms of signal resolution and S/N ratio, as compared to alpha-cyano-4-hydroxycinnamic acid, which was, on the other hand, found to be the only matrix compound that enabled fully automatic mass spectra acquisition. The robustness of the whole sample preparation procedure was then assessed on a set of 25 strains of four Acinetobacter species. Results showed reproducible detection of subtle mass spectral differences between strains belonging to the same species, although they do not confirm the possibility of reliable strain typing.     

One problem of acid rain: Aluminum

In this paper we analyze the equilibrium distribution of different cations M^m+, where m goes from 1 to 3 so that M can be the proton, a calcium, or an aluminum ion, with ligands, ROⁿ⁻, where the pK_a of the acid H_nOR can be small or large. The competition from aluminum for calcium sites can be very considerable in more acidic media. Further considerations show that the problem of acidification is complicated by solid/solution reactions which are not necessarily reversed by reversing the pH changes since the conditions under which the solids dissolve and the final reprecipitation of aluminum are not the same.     

Comparison of single-phase isotropic elastic and fibril-reinforced poroelastic models for indentation of rabbit articular cartilage

Classically, single-phase isotropic elastic (IE) model has been used for in situ or in vivo indentation analysis of articular cartilage. The model significantly simplifies cartilage structure and properties. In this study, we apply a fibril-reinforced poroelastic (FRPE) model for indentation to extract more detailed information on cartilage properties. Specifically, we compare the information from short-term (instantaneous) and long-term (equilibrium) indentations, as described here by IE and FRPE models. Femoral and tibial cartilage from rabbit (age 0–18 months) knees (n=14) were tested using a plane-ended indenter (diameter=0.544 mm). Stepwise creep tests were conducted to equilibrium. Single-phase IE solution for indentation was used to derive instantaneous modulus and equilibrium (Young's) modulus for the samples. The classical and modified Hayes’ solutions were used to derive values for the indentation moduli. In the FRPE model, the indentation behavior was sample-specifically described with three material parameters, i.e. fibril network modulus, non-fibrillar matrix modulus and permeability. The instantaneous and fibril network modulus, and the equilibrium Young's modulus and non-fibrillar matrix modulus showed significant (p<0.01) linear correlations of R²=0.516 and 0.940, respectively (Hayes’ solution) and R²=0.531 and 0.960, respectively (the modified Hayes’ solution). No significant correlations were found between the non-fibrillar matrix modulus and instantaneous moduli or between the fibril network modulus and the equilibrium moduli. These results indicate that the instantaneous indentation modulus (IE model) provides information on tensile stiffness of collagen fibrils in cartilage while the equilibrium modulus (IE model) is a significant measure for stiffness of PG matrix. Thereby, this study highlights the feasibility of a simple indentation analysis.     

Respiratory Chain of Plant Mitochondria: XVIII. Point of Interaction of the Alternate Oxidase with the Respiratory Chain

Oxidation of the respiratory chain carriers of anaerobic, CO-saturated skunk cabbage (Symplocarpus foetidus) mitochondria, by means of an O₂ pulse, proceeds primarily through the cyanide-insensitive alternate oxidase, since the oxidation of cytochromes a and a₃ takes place with a half-time of 3 seconds, corresponding to the rate of dissociation of CO from reduced cytochrome a₃. Ubiquinone and part of the flavoprotein are oxidized within 1 second under these conditions, and this rapid rate of oxidation is strongly inhibited by m-chlorobenzhydroxamic acid (mCLAM), a specific inhibitor of the alternate oxidase of plant mitochondria. The rate of ubiquinone oxidation under these conditions in white potato (Solanum tuberosum) mitochondria, which have no alternate oxidase, is the same as that in skunk cabbage mitochondria treated with mCLAM. Ubiquinone, thus identified as the carrier common to both the cytochrome and alternate oxidase pathways, is linked to the alternate oxidase by a flavoprotein of midpoint potential 50 millivolts more negative with which it is in equilibrium. This arrangement provides a switch for diverting electron transport primarily through the cytochrome pathway under state 3 conditions and primarily through the alternate oxidase pathway under state 4 conditions.     

Interaction of spermine and DNA

The effect of spermine upon the denaturation temperature (T_m) of DNA's of various base compositions has been found to depend upon both the base composition of the DNA and the pH of the solution. Measurement of the hydrogen ion titration curve of spermine as a function of temperature reveals that the net charge of the spermine molecule is undergoing a rapid change with temperature in the range of temperatures at which DNA denatures. Since the value of T_m depends upon base composition, the correlation of the effect of spermine upon T_m with the base composition of the DNA used may be explainable in terms of the changing degree of ionization of spermine. The binding of spermine to native DNA has also been studied by dialysis equilibrium. There is no significant variation either in the number of strongly binding sites or strength of binding with base composition. It is concluded that there is no evidence of correlation between the binding of spermine and the base composition of DNA.     

General ligands in affinity chromatography. Cofactor–substrate elution of enzymes bound to the immobilized nucleotides adenosine 5′-monophosphate and nicotinamide–adenine dinucleotide

1. Two different gels have been prepared suitable for the separation of a number of enzymes, in particular NAD⁺-dependent dehydrogenases, by affinity chromatography. For both the matrix used was Sepharose 4B. For preparation (a), NAD⁺–Sepharose, 6-aminohexanoic acid has been coupled to the gel by the cyanogen bromide method and then NAD⁺ was attached by using dicyclohexylcarbodi-imide; for preparation (b), AMP–Sepharose, N⁶-(6-aminohexyl)-AMP has been coupled directly to cyanogen bromide-activated gel. 2. Affinity columns of both gels retain only the two enzymes when a mixture of bovine serum albumin, lactate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase is applied. Subsequent elution with the cofactor NAD⁺ yields glyceraldehyde 3-phosphate dehydrogenase whereas lactate dehydrogenase is eluted by applying the same molarity of the reduced cofactor. 3. The binding of both glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase to the gel tested, AMP–Sepharose, is strong enough to resist elution by gradients of KCl of up to at least 0.5m. A 0.0–0.15m gradient of the competitive inhibitor salicylate, however, elutes both enzymes efficiently and separately. 4. The elution efficiency of lactate dehydrogenase from AMP–Sepharose has been examined by using a series of eluents under comparable conditions of concentration etc. The approximate relative efficiencies are: 0 (lactate); 0 (lactate+semicarbazide); 0 (0.5mm-NAD⁺); 80 (lactate+NAD⁺); 95 (lactate+semicarbazide+NAD⁺); 100 (0.5mm-NADH). 5. All contaminating lactate dehydrogenase activity can be removed from commercially available crude pyruvate kinase in a single-step procedure by using AMP–Sepharose.     

A test for nucleotide sequence homology

Two macromolecular sequences which have evolved from a common ancestor sequence will tend to include a large number of elements unaffected by replacement mutations in both sequences, as long as the evolutionary rate is not too high or the divergence time is not too great. The positions of corresponding elements may have changed in either daughter sequence due to deletion/insertion mutations involving other sequence elements, but their order can be expected to be the same in both sequences. These sets of correspondences, called matches, may be computed by a recursive algorithm which incorporates constraints on the number of deletion/insertion mutations hypothesized to have occurred. A test is developed which computes the significance of each deletion/insertion hypothesized, based on Monte-Carlo sampling of random sequences with the same base composition as the experimental sequences being tested. Applying the test to 5 S RNAs confirms the relation of Escherichia coli and KB carcinoma 5 S RNAs and establishes the previously undetected homology between Pseudomonas fluorescens and KB 5 S RNAs.     

Patterns and Possible Roles of LINE-1 Methylation Changes in Smoke-Exposed Epithelia

Tobacco smoking and reduced methylation of long interspersed element-1 (LINE-1) are crucial in oral carcinogenesis. 5′UTR of human LINE-1 sequence contains several CpG dinucleotides which are methylated in various proportions (0–100%). Methylation levels of many LINE-1s in cancer were reduced, hypomethylated. The hypomethylation of each LINE-1 locus can promote instability of genome and repress expression of a gene located on that same chromosome. This study investigated if cigarette smoking influences LINE-1 methylation of oral mucosal cells. The methylation of human LINE-1 in clinically normal oral mucosa of current smokers was compared to non-smokers. By using the combined bisulphite restriction analysis, each LINE-1 sequence was categorised into 4 patterns depending on the methylation status and location of the two 18-bp successive CpG from 5′ to 3′ including ^mC^mC, ^uC^uC, ^mC^uC and ^uC^mC. Of these, ^mC and ^uC represent methylated and unmethylated CpG, respectively. The DNA bisulphite sequence demonstrated that most CpGs of ^mC^mC and ^uC^uC were methylated and unmethylated, respectively. Nevertheless, some CpGs of each ^mC^uC or ^uC^mC allele were methylated. Imaging of the digestion products was used to generate %methylation value. No significant difference in the overall LINE-1 methylation level but the differences in percentages of some methylation patterns were discovered. The %^mC^mC and %^uC^uC increased, while the %^mC^uC decreased in current smokers (p = 0.002, 0.015, and <0.0001, respectively). Additionally, the lower %^mC^uC still persisted in persons who had stopped smoking for over 1 year (p = 0.001). The %^mC^uC also decreased in the higher pack-year smokers (p = 0.028). Smoking possibly altered ^mC^uC to ^mC^mC and ^uC^uC forms, and changes ^uC^mC to ^uC^uC forms. In conclusion, smoking changes methylation levels of partial methylated LINE-1s and increased the number of hypo- and hypermethylated loci. These hypomethylated LINE-1s may possess carcinogenesis potential. Moreover, LINE-1 methylation patterns may be useful for monitoring oral carcinogenesis in smokers.