Nucleosomal arrays self‐assemble into supramolecular globular structures lacking 30‐nm fibers

The existence of a 30‐nm fiber as a basic folding unit for DNA packaging has remained a topic of active discussion. Here, we characterize the supramolecular structures formed by reversible Mg²⁺‐dependent self‐association of linear 12‐mer nucleosomal arrays using microscopy and physicochemical approaches. These reconstituted chromatin structures, which we call “oligomers”, are globular throughout all stages of cooperative assembly and range in size from ~50 nm to a maximum diameter of ~1,000 nm. The nucleosomal arrays were packaged within the oligomers as interdigitated 10‐nm fibers, rather than folded 30‐nm structures. Linker DNA was freely accessible to micrococcal nuclease, although the oligomers remained partially intact after linker DNA digestion. The organization of chromosomal fibers in human nuclei in situ was stabilized by 1 mM MgCl₂, but became disrupted in the absence of MgCl₂, conditions that also dissociated the oligomers in vitro. These results indicate that a 10‐nm array of nucleosomes has the intrinsic ability to self‐assemble into large chromatin globules stabilized by nucleosome–nucleosome interactions, and suggest that the oligomers are a good in vitro model for investigating the structure and organization of interphase chromosomes.     

Comparison of the structure of ribosomal 5S RNA from E. coli and from rat liver using X-ray scattering and dynamic light scattering

The structures of eukaryotic ribosomal 5S RNA from rat liver and of prokaryotic 5S RNA from E. coli (A-conformer) have been investigated by scattering methods. For both molecules, a molar mass of 44,500±4,000 was determined from small angle X-ray scattering as well as from dynamic light scattering. The shape parameters of the two rRNAs, volume V _c, surface O _c, radius of gyration R _s, maximum dimension of the molecule L, thickness D, and cross section radius of gyration R _sq, agree within the experimental error limits. The mean values are V _c=57±3 nm³, O _c=165±10 nm², R _s=3.37±0.05 nm, L=10.8±0.7 nm, D=1.57±0.07 nm, R _sa=0.92±0.01 nm.Identical structures for the E. coli 5S rRNA and the rat liver 5S rRNA at a resolution of 1 nm can be deduced from this agreement and from the comparison of experimental X-ray scattering curves and of experimental electron distance distribution function. The flat shape model derived for prokaryotic and eukaryotic 5S rRNA shows a compact region and two protruding arms. Double helical stems are eleven-fold helices with a mean base pair distance of 0.28 nm. Combining the shape information obtained from X-ray scattering with the information about the frictional behaviour of the molecules, deduced from the diffusion coefficients D _20,w ⁰ =(5.9±0.2)·10^-7 cm²s^-1 and (6.2±0.2)·10^-7 cm²s^-1 for rat liver 5S rRNA and E. coli 5S rRNA, respectively, a solvation shell of about 0.3 nm thickness around both molecules is determined. This structural similarity and the consensus secondary structure pattern derived from comparative sequence analyses suggest that all 5S rRNAs may indeed have conserved essentially the same type of folding of their polynucleotide strands during evolution, despite having very different sequences.     

Quantitative analysis of calcium gradients and activity in growing pollen tubes ofLilium longiflorum

Summary Pollen tubes ofLilium longiflorum were loaded with quin-2 to determine the cytoplasmic free calcium. Quin-2-fluorescence was detected at 500 nm with alternating excitation at 340 nm and 360 nm. The calcium²⁺-concentration was obtained using the intensity ratio R=I₃₄₀/I₃₆₀. The analysis exhibits a [Ca²⁺] of nearly 10^–7mol·l^–1 in the tip region and about 2·10^–8 mol·l^–1at the tube base, near the pollen grain. The data give evidence for the existence of a continuous gradient of free calcium within growing pollen tubes of various length.     

Cesium,Na+-K+ pump and pacemaker potential in cardiac purkinje fibers

The mechanisms of the hyperpolarizing and depolarizing actions of cesium were studied in cardiac Purkinje fibers perfused in vitro by means of a microelectrode technique under conditions that modify either the Na⁺-K⁺ pump activity or I_f. Cs⁺ (2 mM) inconsistently increased and then decreased the maximum diastolic potential (MDP); and markedly decreased diastolic depolarization (DD). Increase and decrease in MDP persisted in fibers driven at fast rate (no diastolic interval and no activation of I_f). In quiescent fibers, Cs⁺ caused a transient hyperpolarization during which elicited action potentials were followed by a markedly decreased undershoot and a much reduced DD. In fibers depolarized at the plateau in zero [K⁺]_o (no I_f), Cs⁺ induced a persistent hyperpolarization. In 2 mM [K⁺]_o, Cs⁺ reduced the undershoot and suppressed spontaneous activity by hyperpolarizing and thus preventing the attainment of the threshold. In 7 mM [K⁺]_o, DD and undershoot were smaller and Cs⁺ reduced them. In 7 and 10 mM [K⁺]_o, Cs⁺ caused a small inconsistent hyperpolarization and a net depolarization in quiescent fibers; and decreased MDP in driven fibers. In the presence of strophanthidin, Cs⁺ hyperpolarized less. Increasing [Cs⁺]_o to 4, 8 and 16 mM gradually hyperpolarized less, depolarized more and abolished the undershoot. We conclude that in Purkinje fibers Cs⁺ hyperpolarizes the membrane by stimulating the activity of the electrogenic Na⁺-K⁺ pump (and not by suppressing I_f); and blocks the pacemaker potential by blocking the undershoot, consistent with a Cs⁺ block of a potassium pacemaker current.     

Li+ counterion self-diffusion in ordered DNA

The anisotropic self-diffusion coefficient of ⁷Li⁺ (I = 3/2) counterions has been studied in hydrated, macroscopically oriented Li-(B)DNA fibers at relatively high water contents, corresponding to approximate DNA-DNA helix axis distances of 22–35 Å, using the pulsed field gradient hmr spin-echo method. Self-diffusion coefficients parallel (D_∥) and perpendicular (D_?) to the DNA helix axis increase with increasing salt content and with increasing DNA-DNA helix axis distance. The observed anisotropy D_∥/D_? decreases from 1.6 to 1.2 with the DNA-DNA separation increasing from 22 to 35 Å in the salt-free sample. This result can be understood by the obstruction effect caused by the DNA molecules themselves. The values of the Li⁺ self-diffusion coefficients in the most water-rich system with no added salt (corresponding to an approximate distance of 35 Å between the DNA helix axes) were D_∥ ～ 1.15 × 10^?10 m² s^?1 and D_? ～ 0.98 × 10^?10 m² s^?1, compared to 9.14 × 10^?10 m² s^?1 for the diffusion of Li⁺ in an aqueous solution of LiCl (～ 2.1M). The possible occurrence of restriction effects in the DNA fibers have also been studied by determining the self-diffusion coefficient at different effective diffusion times. The self-diffusion coefficient of Li⁺ in the sample with the largest DNA-DNA helix axis distance seems to be independent of the effective diffusion time, which indicates that the lithium ions are not trapped within impermeable barriers. The possibility of diffusion through permeable barriers has also been investigated, and is discussed. © 1994 John Wiley & Sons, Inc.     

The fine structure of euchromatin and centromeric heterochromatin in Tenebrio molitor chromosomes

The fine structure of constitutive heterochromatin and euchromatin was compared in electron microscope whole-mount preparations of Tenebrio molitor (Insecta, Coleoptera) spermatocyte nuclei. Tenebrio molitor pachytene chromosomes display extended segments of centromeric heterochromatin and thus are especially suitable for this purpose. When nuclei were incubated in solutions containing different concentrations of NaCl or of MgCl₂, two levels of chromatin fine structures were observed in the euchromatic segments: nucleosome fibers (0.1 mM–20 mM NaCl) and supranucleosomal fibers with 28 nm in diameter (40 mM–100 mM NaCl, 0.2 mM–1.0 mM MgCl₂). The fine structure in the heterochromatic segments was the same as that in the euchromatic segments in all NaCl concentrations and in MgCl₂ concentrations up to 0.4 mM. In higher MgCl₂ concentrations the heterochromatin remained more compact than the euchromatin and consisted of 37-nm-thick fibers in 0.6 mM MgCl₂ and of 65-nm-thick fibers in 1.0 mM MgCl₂. After the 37-nm and the 65-nm fibers had been dispersed in Mg²⁺-free solutions they could be recondensed by incubation in 0.6 mM and 1.0 mM MgCl₂, respectively. It is concluded that a Mg²⁺-sensitive component of the heterochromatin is responsible for the folding of the nucleosome chain to heterochromatin-specific supranucleosomal structures.     

The cole-moore effect in nodal membrane of the frogRana ridibunda: Evidence for fast and slow potassium channels

Summary The K conductance (g _K) kinetics were studied in voltage-clamped frog nodes (Rana ridibunda) in double-pulse experiments. The Cole-Moore translation forg _K–t curves associated with different initial potentials (E) was only observed with a small percentage of fibers. The absence of the translation was found to be caused by the involvement of an additional, slow,g _K component. This component cannot be attributed to a multiple-state performance of the K channel. It can only be accounted for by a separate, slow K channel, the fast channel being the same as then ⁴ K channel inR. pipiens.The slow K channel is characterized by weaker sensitivity to TEA, smaller density, weaker potential (E) dependence, and somewhat more negativeE range of activation than the fast K channel. According to characteristics of the slow K system, three types of fibers were found. In Type I fibers (most numerous) the slow K channel behaves as ann ⁴ HH channel. In Type II fibers (the second largest group found) the slow K channel obeys the HH kinetics within a certainE range only; beyond this range the exponential decline of the slowg _K component is preceded by anE-dependent delay, its kinetics after the delay being the same as those in Type I fibers. In Type III fibers (rare) the slow K channel is lacking, and it is only in these fibers that the Cole-Moore translation of the measuredg _K–t curves can be observed directly.The physiological role of the fast and slow K channel in amphibian nerves is briefly discussed.     

Functionalization of poly(amidoamine) dendrimer‐based nano‐architectures using a naphthalimide derivative and their fluorescent,dyeing and antimicrobial properties on wool fibers

Novel naphthalimide–poly(amidoamine) dendrimer fluorescent dyes were synthesized, and their structures were identified and confirmed using different characterization methods such as Fourier transform infrared, ¹H NMR, ¹³C NMR, differential scanning calorimetry, elemental analysis and UV–vis spectroscopy. The spectrophotometric studies demonstrated absorption maxima (λ_max) and extinction coefficient (ε_max) values in the ranges of 429–438 nm and 25,635–88,618 L/mol/cm, respectively. The dyeing, fastness and antimicrobial properties of dyed wool fibers were examined. Colorimetric measurements demonstrated a greenish‐yellow hue with remarkable fluorescence intensity on dyed wool. Although the fastness properties of naphthalimide dye on wool fibers were poor/moderate, color fastness was appreciably improved through modification of the dye using dendrimers. The results revealed that the newly synthesized dyes are potent antimicrobial agents on wool fibers. Overall, it was deduced that poly(amidoamine) (PAMAM) dendrimers could be exploited as a promising tool in tailoring the different properties of naphthalimide dyes, being suitable for dyeing and antimicrobial finishing agents for wool fibers. Copyright © 2015 John Wiley & Sons, Ltd.     

Muscarinic activation causes biphasic inotropic response and decreases cellular Na+ activity in canine cardiac purkinje fibers

In this study, the effects of carbachol (CCh) on twitch tension, intracellular Na⁺ activity (a _Na ⁱ ), and action potential were simultaneously measured in canine cardiac Purkinje fibers in order to examine the regulation of inotropy through muscarinic receptors and its relation to a _Na ⁱ . In fibers driven at 1 Hz, CCh (10 µM) initially and transiently decreased and then increased the twitch tension by 36±8%. The action potential showed a significant elevation of the plateau and a significant shortening of the duration at 90% repolarization (APD₉₀), from 403±7 to 389±7 ms. The a _Na ⁱ decreased from 7.4±0.4 to 6.7±0.3 mM (n=23, p<0.05). Atropine (1 µM) decreased the twitch tension by 21±6% (n=7, p<0.05) without significant effects on the action potential and a _Na ⁱ , and inhibited the effects of CCh. Cs⁺ (20 mM) increased the plateau height and APD₉₀, enhanced the twitch tension by 66±24%, but decreased a _Na ⁱ from 7.3±0.3 to 6.3±0.4 mM (n=6, p<0.05). In the presence of 20 mM Cs⁺, some fibers generated slow responses. The addition of 10 µM CCh further increased the twitch tension and APD₉₀, and decreased a _Na ⁱ from 6.3±0.4 to 5.3±0.3 mM. Ouabain (0.3 µM) increased the twitch tension and a _Na ⁱ , and inhibited the CCh-induced decrease of a _Na ⁱ . In the presence of ouabain, 20 mM Cs⁺ depolarized the fiber and generated slow responses with a decreased a _Na ⁱ . The addition of 10 µM CCh enhanced the slow action potential, and increased a _Na ⁱ although there was a transient decrease during early exposure. These results suggest that activation of muscarinic receptors in canine Purkinje fibers results in an enhancement of the Na⁺-K⁺ pump activity and a biphasic inotropic response, probably via different receptor subtypes. The inhibitory effect, most likely through M₂ receptors, is associated with the activation of K⁺ channels. The stimulatory effect, on the other hand, is probably due to the action on the M₁ receptors, resulting in increases in Ca²⁺ currents.     

Axon transport of steroid hormones via spinal root fibers in old rats

Labelled steroid hormones,³H-hydrocortisone and¹⁴C-testosterone, being injected in the gray matter of theL ₅−L ₆ spinal cord segments were shown to be transported via ventral and dorsal root fibers (antero- and retrograde directions, respectively) of old (25 to 28 months) rats with a lower velocity than in adult young (6 to 11 months) animals. The averaged maximum velocities of axon transport (AT) through the ventral and dorsal roots were: for³H-hydrocortisone, 756±63 and 738±46 mm per day, and for¹⁴H-testosterone, 624±54 and 608±80 mm per day, respectively. Therefore, in old rats the AT velocities for³H-hydrocortisone and¹⁴C-testosterone were about four and seven-eight times lower than those in adult rats. In the course of anterograde, AT through the ventral roots in old rats the inclusion of³H-hydrocortisone is sharply suppressed (by more than an order of magnitude), as compared with than in adult animals. The doses of non-labelled steroid hormones within a 10⁻⁷ – 10⁻⁶ range, injected into the lumbar spinal segments, resulted in hyperpolarization of muscle fibers of themm. gastrocnemius anddeltoideus, but this phenomenon developed in old rats much later than in adult rats. It is obvious that AT of steroid hormones can be considered one of the mechanisms of their effects on the tissue of an organism, and this mechanism undergoes extremely intensive modifications with aging.     

Structure of native proteoglycan aggregates from chick limb-bud chondrocytes

Laser light-scattering has been used to investigate the size of native proteoglycan aggregates (PGA-aA1) from day-8 chick limb-bud chondrocyte cultures isolated under associative extraction and purification conditions in 0.4M guanidinium chloride (GdnHCl) solution. Dynamic light-scattering measurements yielded a hydrodynamic radius, R_s, of 244 ± 10 nm for PGA-aA1 in 0.4M GdnHCl, and a weight-average molecular weight (M _w) of 150 ± 50 × 10⁶ was obtained from a Zimm plot. Disaggregation in 4.0M GdnHCl aqueous solution yielded proteoglycan subunits (PGS) with R_s = 39 ± 2 nm, M _w = 1.6 ± 0.3 × 10⁶, which reassembled in 0.4M GdnHCl to form “reconstituted native” aggregates (PGA-raA1) with R_s = 121 ± 6 nm, M _w = 17 ± 3 × 10⁶. A second specimen of PGA-aA1 had R_s = 192 ± 10 nm, M _w = 100 ± 10 × 10⁶. The latter value was estimated from an empirical relationship between M _w and R_s. After dissociation, this specimen reassembled to form PGA-raA1 with R_s = 85 ± 5 nm, M _w = 12 ± 1 × 10⁶. These data are compared with those for a specimen of reconstituted aggregate (PGA-A1) that had been extracted under dissociative conditions and then reaggregated by dialysis to 0.4M GdnHCl aqueous solution, for which R_s = 138 ± 9 nm, M _w = 45 ± 8 × 10⁶. From these values, we have calculated the weight-average number of subunits per aggregate N_w: 111 for PGA-aA1 and 12 for raA1 (70 and 7 for the second PGA-aA1 and PGA-raA1 specimen, respectively) as compared to 32 for PGA-A1. The numbers of subunits per aggregate were also determined from electron micrographs of spread specimens. The latter results show the same trends as those obtained by light scattering, but lead in each case to lower numbers of subunits per aggregate. These data demonstrate conclusively that PGA samples exhibit a higher degree of aggregation in solution than visualized in typical electron microscopy (EM) preparations, probably due to disaggregation during EM specimen preparation. Since N_w determined both by light scattering (LS) and by EM are larger for native versus reconstituted aggregate samples, our data point to a more compact aggregation of subunits along the hyaluronic acid (HA) chains in the former.     

Efficiency of KrCl excilamp (222 nm) for inactivation of bacteria in suspension

Aims: To examine the killing efficiency of UV KrCl excilamp against Gram‐positive and Gram‐negative bacteria. Methods and Results: Vegetative cells of Bacillus cereus, Bacillus subtilis, Escherichia coli O157:H7, Staphylococcus aureus and Streptococcus pyogenes at initial populations from 10² to 10⁷ colony‐forming units (CFU) ml^?1 were treated by KrCl excilamp in sterile Ringer’s solution with and without H₂O₂. The number of viable cells was determined using spread plating techniques and nutrient agar method with subsequent incubation at 28°C or 37°C for 24 h. At estimated populations of 10²–10⁵ CFU ml^?1E. coli O157:H7 and Staph. aureus were the most sensitive and showed 100% disinfection within 15 s (29·2 mJ cm^?2). Bacillus subtilis was more sensitive to UV treatment than B. cereus. The UV/H₂O₂ inactivation rate coefficients within this population range were two times higher than those observed for UV treatment alone. No effect of H₂O₂ was observed at 10⁷ CFU ml^?1 for Bacillus sp. and Strep. pyogenes. Conclusions: The narrow‐band UV radiation at 222 nm was effective in the rapid disinfection of bacteria in aqueous suspensions. Significance and Impact of the Study: KrCl excilamps represent UV sources which can be applied for disinfection of drinking water in advanced oxidation processes.     

Luminescence properties of novel deep‐red‐emitting Ca3Al2Ge2O10:Cr3+ phosphors

Ca₃Al₂Ge₂O₁₀:Cr³⁺ phosphors were prepared by a high‐temperature solid‐state method, and their luminescence properties were investigated. Under excitation at 550 nm, Ca₃Al₂Ge₂O₁₀:Cr³⁺ phosphors exhibited a broad red emission band at 697 nm in the range 650–750 nm that was caused by the ²E→⁴A₂ transition of Cr³⁺. For the 697 nm emission peak, emission intensity reached a maximum at x = 0.07, and there was concentration quenching of Cr³⁺ in Ca₃Al₂Ge₂O₁₀; the corresponding concentration quenching mechanism was analysed. Under excitation at 262 nm, the Ca₃Al₂Ge₂O₁₀:Cr³⁺ phosphor showed a weakly broad emission band in the range 350–600 nm that was caused by intrinsic defects (V′′_Ca and V′′_O). Copyright © 2016 John Wiley & Sons, Ltd.     

Multi-color luminescence of Sm3+-doped Na2YMg2V3O12 phosphor potentially applicable in white-LEDs

A novel multi-color emitting Na₂YMg₂V₃O₁₂:Sm³⁺ phosphor was synthesized using a solid-state reaction, and its crystal structure, luminescence properties, and thermal stability were studied. Charge transfer within the (VO₄)³⁻ groups in the Na₂YMg₂V₃O₁₂ host led to a broad emission band between 400 and 700 nm, with a maximum at 530 nm. The Na₂Y_1−xMg₂V₃O₁₂:xSm³⁺ phosphors exhibited a multi-color emission band under 365 nm near-ultraviolet (near-UV) light, consisting of the green emission of the (VO₄)³⁻ groups and sharp emission peaks at 570 nm (yellow), 618 nm (orange), 657 nm (red), and 714 nm (deep red) of Sm³⁺ ions. The optimal doping concentration of Sm³⁺ ions was found to be 0.05 mol%, and the dipole–dipole (d–d) interaction was primarily responsible for the concentration quenching phenomenon. Using the acquired Na₂YMg₂V₃O₁₂:Sm³⁺ phosphors, commercial BaMgAl₁₀O₁₇:Eu²⁺ blue phosphor, and a near-UV light-emitting diode (LED) chip, a white-LED lamp was designed and packaged. It produced bright neutral white light, manifesting a CIE coordinate of (0.314, 0.373), a color rendering index (CRI) of 84.9, and a correlated color temperature (CCT) of 6377 K. These findings indicate the potential of Na₂YMg₂V₃O₁₂:Sm³⁺ phosphor to be used as a multi-color component for solid-state illumination.     

A ratiometric fluorescence strategy based on copper nanoclusters/carbon dots for sensitive detection of doxorubicin

Sensitive detection of doxorubicin (DOX) is critical for clinical theranostics. A novel ratiometric fluorescence strategy based on the inner filter effect (IFE) has been established for the sensitive detection of DOX by designing a ratiometric fluorescence probe. In the presence of DOX, the fluorescence intensity of copper nanoclusters (CuNCs) at 485 nm decreases, and the fluorescence intensity of carbon dots at 560 nm increases. Therefore, DOX can be quantitatively detected by measuring the ratio of the fluorescence intensities at 560 and 485 nm (F₅₆₀/F₄₈₅). The F₅₆₀/F₄₈₅ ratio exhibits a linear correlation with the DOX concentration in the range from 1.0 × 10⁻⁸ M to 1.0 × 10⁻⁴ M with the detection limit of 3.7 nM. Furthermore, this method was also successfully applied to the analysis of DOX in human plasma samples, affording an effective platform for drug safety management.     

An investigation of the effects of external acidification on sodium transport,internal pH and membrane potential in barnacle muscle fibers

Summary Radiosodium efflux from barnacle muscle fibers is a function of pH _e , the threshold pH _e for stimulation of Na efflux into HCO ₃ ^– -artificial sea water (ASW) being 6.8 and the fixed thresholdpCO₂ (in an open CO₂ system) being approximately 30 mm Hg. Acidification of ASW containing non-HCO ₃ ^– buffer is without effect on the Na efflux. The Na efflux following stimulation by reducing the pH of 10mM HCO ₃ ^– -ASW from 7.8 to 6.3 is reduced by 17.3% as the result of microinjecting 100mM EGTA, and increased by 32.6% as the result of microinjecting 0.5M ATP. The Na efflux into K-free HCO ₃ ^– -ASW is markedly stimulated by external acidification. Ouabain-poisoned fibers are more responsive to a low pH _e than unpoisoned fibers. Applying the 2-¹⁴C-DMO technique, it is found that fibers bathed in 10mM HCO ₃ ^– -ASW at pH 7.8 have an internal pH of 7.09±0.106 (mean±SD), whereas fibers bathed in 25mM TRIS-ASW at pH 7.8 have a pH _i of 7.28±0.112. The relationship between pH _i and pH _e as external pH is varied by adding H⁺ is linear. Measurements of the resting membrane potential indicate that external acidification in the presence of HCO ₃ ^– as buffer is accompanied by a fall inE _m , the threshold pH _e being 7.3 both at 24 and 0°C. This sensitivity amounts to 8.2 mV per pH unit (at 24°C) over a wide range of pH _e . Membrane resistance following external acidification remains unchanged. Microinjection of the protein inhibitor of Walsh before external acidification fails to stop depolarization from occurring. Cooling to 0°C also fails to abolish depolarization following acidification. Whereas external ouabain and ethacrynic acid reduceE _m in the absence or presence of acidification, DPH hyperpolarizes the membrane or arrests depolarization both at 24 and 0°C. This effect of DPH at 0°C is seen in the absence or presence of acidification. It is suggested that depolarization following acidification of a HCO ₃ ^– -containing medium is due to activation of a Cl^–-and/or HCO ₃ ^– -pump and that ouabain and ethacrynic acid reducesE _m by abolishing uncoupled Na transport.     

Stimulation by high external potassium of the sodium efflux in barnacle muscle fibers

Summary Single barnacle muscle fibers fromBalanus nubilus were used to study the effect of elevated external potassium concentration, [K] _o , on Na efflux, membrane potential, and cyclic nucleotide levels. Elevation of [K] _o causes a prompt, transient stimulation of the ouabain-insensitive Na efflux. The minimal effective concentrations is 20mm. The membrane potential of ouabain-treated fibers bathed in 10mm Ca²⁺ artificial seawater (ASW) or in Ca²⁺-free ASW decreases approximately linearly with increasing logarithm of [K] _o . The slope of the plot is slightly steeper for fibers bathed in Ca²⁺-free ASW. The magnitude of the stimulatory response of the ouabain-insensitive Na efflux to 100mmK _o depends on the external Na⁺ and Ca²⁺ concentrations, as well as on external pH, but is independent of external Mg²⁺ concentration. External application of 10^–4 m verapamil virtually abolishes the response of the Na efflux to subsequent K-depolarization. Stabilization of myoplasmic-free Ca²⁺ by injection of 250mm EGTA before exposure of the fiber to 100mm K _o leads to 60% reduction in the magnitude of the stimulation. Pre-injection of a pure inhibitor of cyclic AMP-dependent protein kinase reduces the response of the Na efflux to 100mm K _o by 50%. Increasing intracellular ATP, by injection of 0.5m ATP-Na₂ before elevation of [K] _o , fails to prolong the duration of the stimulation of the Na efflux. Exposure of ouabain-treated, cannulated fibers to 100mm K _o for time periods ranging from 30 sec to 10 min causes a small (60%), but significant, increase in the intracellular content of cyclic AMP with little change in the cyclic GMP level. These results are compatible with the view that the stimulatory response of the ouabain-insensitive Na efflux to high K _o is largely due to a fall in myoplasmicpCa resulting from activation of voltage-dependent Ca²⁺ channels and that an accompanying rise in internal cAMP accounts for a portion of this response.     

Measurement of diffusion coefficient and electrophoretic mobility with a quasielastic light-scattering–band-electrophoresis apparatus

We have constructed an apparatus for the simultaneous measurement of electrophoretic mobility, μ, and diffusion coefficient, D, of macromolecules and cells. It combines band electrophoresis in a vertical, sucrose-gradient stabilized column, with quasielastic laser light-scattering determination of the diffusion coefficient of the species within the band. The entire electrophoresis cell is scanned through the laser beam of the quasielastic laser light-scattering apparatus by a vertical translation stage. Total intensity light-scattering measurement at each point in the cell gives the macromolecular concentration at that point. Solvent viscosity and electrical potential are measured at each point in the cell. Application of this apparatus to resealed red blood cell ghosts and to bovine hemoglobin indicates that measurements of field, viscosity, and migration distance are reliable, and that electroosmosis is insignificant. Application to T4D bacteriophage gives μ_20,w = (?1.05 ± 0.05) × 10^?4 cm²/V sec and D_20,w = (3.35 ± 0.10) × 10^?8 cm²/sec for fiberless particles, and μ_20,w = ?(0.59 ± 0.03) × 10^?4 cm²/V sec and D_20,w = (2.86 ± 0.09) × 10^?8 cm²/sec for whole phage with 6 fibers. Approximate analysis of these results with the Henry electrophoresis theory for spheres in dicates that each fiber contributes about 193 positive charges to the phage particle, compared with 327 from amino-acid analysis. The advantages and disadvantages of this apparatus, relative to conventional electrophoresis and to electrophoretic light scattering, are discussed.     

Titanium dioxide nanoparticles preferentially bind in subdomains IB,IIA of HSA and minor groove of DNA

Titanium dioxide nanoparticles (TiO₂-NPs) interaction with human serum albumin (HSA) and DNA was studied by UV–visible spectroscopy, spectrofluorescence, circular dichroism (CD), and transmission electron microscopy (TEM) to analyze the binding parameters and protein corona formation. TEM revealed protein corona formation on TiO₂-NPs surface due to adsorption of HSA. Intrinsic fluorescence quenching data suggested significant binding of TiO₂-NPs (avg. size 14.0 nm) with HSA. The Stern–Volmer constant (K_sv) was determined to be 7.6 × 10² M^?1 (r² = 0.98), whereas the binding constant (K_a) and number of binding sites (n) were assessed to be 5.82 × 10² M^?1 and 0.97, respectively. Synchronous fluorescence revealed an apparent decrease in fluorescence intensity with a red shift of 2 nm at Δλ = 15 nm and Δλ = 60 nm. UV–visible analysis also provided the binding constant values for TiO₂-NPs–HSA and TiO₂-NPs-DNA complexes as 2.8 × 10² M^?1 and 5.4 × 10³ M^?1. The CD data demonstrated loss in α-helicity of HSA and transformation into β-sheet, suggesting structural alterations by TiO₂-NPs. The docking analysis of TiO₂-NPs with HSA revealed its preferential binding with aromatic and non-aromatic amino acids in subdomain IIA and IB hydrophobic cavity of HSA. Also, the TiO₂-NPs docking revealed the selective binding with A-T bases in minor groove of DNA.     

Caffeine inhibition of calcium accumulation by the sarcoplasmic reticulum in mammalian skinned fibers

Summary Oxalate-supported Ca accumulation by the sarcoplasmic reticulum (SR) of chemically skinned mammalian skeletal muscle fibers is activated by MgATP and Ca²⁺ and partially inhibited by caffeine. Inhibition by caffeine is greatest when Ca²⁺ exceeds 0.3 to 0.4 m, when free ATP exceeds 0.8 to 1mm, and when the inhibitor is present from the beginning of the loading period rather than when it is added after Ca oxalate has already begun to precipitate within the SR. Under the most favorable combination of these conditions, this effect of caffeine is maximal at 2.5 to 5mm and is half-maximal at approximately 0.5mm. For a given concentration of caffeine, inhibition decreases to one-half of its maximum value when free ATP is reduced to 0.2 to 0.3mm. Varying free Mg²⁺ (0.1 to 2mm) or MgATP (0.03 to 10mm) has no effect on inhibition. Average residual uptake rates in the presence of 5mm caffeine atpCa 6.4 range from 32 to 70% of the control rates in fibers from different animals. The extent of inhibition in whole-muscle homogenates is similar to that observed in skinned fibers, but further purification of SR membranes by differential centrifugation reduces their ability to respond to caffeine. In skinned fibers, caffeine does not alter the Ca²⁺ concentration dependence of Ca uptake (K _0.5, 0.5 to 0.8 m; Hilln, 1.5 to 2.1). Reductions in rate due to caffeine are accompanied by proportional reductions in maximum capacity of the fibers, and this configuration can be mimicked by treating fibers with the ionophore A23187. Caffeine induces a sustained release of Ca from fibers loaded with Ca oxalate. However, caffeine-induced Ca release is transient when fibers are loaded without oxalate. The effects of caffeine on rate and capacity of Ca uptake as well as the sustained and transient effects on uptake and release observed under different conditions can be accounted for by a single mode of action of caffeine: it increases Ca permeability in a limited population of SR membranes, and these membranes coexist with a population of caffeine-insensitive membranes within the same fiber.