Iodination of nucleosomes at low ionic strength: conformational changes in H4 and stabilization by H1.

Radioactive iodine has been used to probe the relative reactivities of nucleosomal H4 tyrosine residues under various conditions of subphysiological ionic strength. We observe that tyrosine 72 of H4, which is not reactive over the range 20-150 mM NaCl, becomes the predominant site of iodination within H4 when nucleosomes are subjected to conditions of very low ionic strength. Conversely, the other H4 tyrosine residues, which are reactive within nucleosomes in solutions of moderate ionic strength (20-150 mM NaCl), become nonreactive when the ionic strength is reduced. This "flip-flop" in the H4 iodination pattern is the manifestation of a reversible nucleosomal conformational change. A method is presented which enables the conformational status of H4 in nucleosomes to be determined by simply electrophoresing the histones on a Triton gel after probing nucleosomes with labeled iodine. Using this technique, we demonstrate that the presence of H1 on one side of the nucleosome stabilizes a histone core domain on the other side so that all four tyrosines of H4 are maintained in their physiological ionic strength conformation even under conditions of no added salt.     

Ionic strength and the polyvalent cation receptor of shark rectal gland and artery

The dogfish shark Squalus acanthias regulates plasma osmolality and extracellular volume by secreting a fluid from its rectal gland which has a higher NaCl and lower urea concentration than plasma. We have previously identified the presence of a calcium-sensing receptor or polyvalent cation sensing receptor (CaSR) on vascular smooth muscle of the rectal gland artery (RGA) and rectal gland tubules (RGT). Activity of the CaSR is influenced by changes in ionic strength. This led us to speculate that the ingestion of invertebrate sea animals increased plasma ionic strength, resulting in inhibition of the receptor, relaxation of RGA, and reversal of inhibition of chloride secretion by the RGT. In contrast, ingestion of fish could diminish ionic strength and have the opposite effect. To study the effect of changes in extracellular ionic strength, shark Ringers solutions were adjusted to three different ionic strengths with NaCl, but the osmolarities were kept constant by varying the concentration of urea. High ionic strength inhibited and low ionic strength enhanced the response to increasing external Ca2+ from 2.5 to 4.7 mM in RGT. The increase in cytosolic Ca2+ ([Ca2+]i) of cells in low, normal, and high ionic strength Ringers solution was 344 +/- 60, 201 +/- 26, and 114 +/- 15 nmol/L, respectively. The [Ca2+]i responses of RGA to external Ca2+ in Ringers of three different ionic strengths were 323 +/- 43, 231 +/- 14, and 56 +/- 11 nmol/L, respectively. Activation of the CaSR by spermine was reduced by more than 50% by high ionic strength in both RGT and RGA. Whether the small changes in shark plasma ionic strength that occur after a shark ingests marine animals with lower and higher ionic strength modulates salt secretion by the rectal gland is not yet known.     

Soil salinity delays germination and limits growth of hyphae from propagules of arbuscular mycorrhizal fungi

Colonisation of plant roots by some arbuscular mycorrhizal (AM) fungi is reduced in the presence of sodium chloride (NaCl), probably due to a direct effect of NaCl on the fungi. However, there appear to be differences between the fungi in their ability to colonise plants in the presence of NaCl. This experiment tested the hypothesis that propagules of different isolates and species of AM fungi from saline and nonsaline soils would differ in their ability to germinate and grow in the presence of NaCl in the soil solution. Spores or pieces of root colonised by a range of AM fungi were incubated between filters buried in soil to which NaCl had been added at concentrations of 0, 150 or 300 mM in the soil solution. At regular intervals, filters were removed from the soil and both the percentage of propagules which had germinated and the length of proliferating hyphae were determined. Germination of spores of AM fungi studied was delayed in the presence of NaCl, but the fungi differed in the extent to which germination was inhibited. Two isolates of Scutellospora calospora reached maximum germination in 300 mM NaCl, but neither of two isolates of Acaulospora laevis germinated in the presence of NaCl. Germination of spores of the other fungi, including some isolated from saline soil, fell between these extremes. For some fungi, the specific rate of hyphal extension was reduced by NaCl. For others, the specific rate of growth was similar in the presence of NaCl to that in the control treatment, but overall production of hyphae was reduced in the NaCl treatments because germination was reduced.     

Characterization of the Hantaan nucleocapsid protein-ribonucleic acid interaction

The nucleocapsid (N) protein functions in hantavirus replication through its interactions with the viral genomic and antigenomic RNAs. To address the biological functions of the N protein, it was critical to first define this binding interaction. The dissociation constant, K(d), for the interaction of the Hantaan virus (HTNV) N protein and its genomic S segment (vRNA) was measured under several solution conditions. Overall, increasing the NaCl and Mg(2+) in these binding reactions had little impact on the K(d). However, the HTNV N protein showed an enhanced specificity for HTNV vRNA as compared with the S segment open reading frame RNA or a nonviral RNA with increasing ionic strength and the presence of Mg(2+). In contrast, the assembly of Sin Nombre virus N protein-HTNV vRNA complexes was inhibited by the presence of Mg(2+) or an increase in the ionic strength. The K(d) values for HTNV and Sin Nombre virus N proteins were nearly identical for the S segment open reading frame RNA, showing weak affinity over several binding reaction conditions. Our data suggest a model in which specific recognition of the HTNV vRNA by the HTNV N protein resides in the noncoding regions of the HTNV vRNA.     

Transport studies with brush border membrane vesicles: choice of experimental parameters

1. We have studied different parameters, in their effects on a transport system chosen as a model: the Na+-phosphate symporter of the renal brush border membrane. 2. Ionic strength was found to be a critical factor in the retention capacity of the filter. 3. When high ionic strength solutions containing 150 mM NaCl or KCl were used, less than 8% of the membrane proteins were lost through filtration. 4. Lowering the ionic strength by replacing NaCl or KCl by 300 mM mannitol, however, caused a 52% loss of protein. 5. Addition of 15 mM NaCl to this low ionic strength solution was sufficient to restore full retention of the vesicles by the filter. 6. The presence of arsenate, a competitive inhibitor, in the stop solution did not improve the retention of phosphate by the vesicles in high ionic strength media, but caused a pronounced temperature dependent loss of the vesicle content, as a function of time of incubation in low ionic strength solutions. 7. Addition of 5 mM phosphate in the stop solution caused a 31 and 37% loss for KCl and NaCl stop solutions, respectively, while no effect was observed for the mannitol stop solution. 8. The presence of HgCl2 gave a 32% stimulation for the mannitol solution and a 35 or 22% inhibition for the KCl or NaCl solutions. 9. Addition of NaCl in the stop solution caused an overaccumulation of 75%, after 60 sec of incubation at 25 degrees C. 10. Phosphate transport by renal vesicles is thus highly affected by the composition of the stop solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

外源海藻糖对小麦幼苗耐盐性的影响

以盐敏感小麦品种鲁麦15为材料,分别用完全Hoagland营养液、150mmol／L NaCl和150mmol／L NaCl 10mmol／L海藻糖处理小麦幼苗,测定小麦幼苗生长、离子含量、根系质膜H^ -ATPase、SOD活性、MDA含量等指标,旨在探讨外源海藻糖在抗盐性中的作用。结果表明：外源海藻糖可明显缓解盐胁迫对小麦幼苗生长的抑制作用;明显提高NaCl胁迫条件下小麦幼苗叶片中K^ 的含量,降低Na^ 的含量,降低其Na^ ／K^ ;提高NaCl胁迫条件下小麦幼苗SOD活性,降低MDA的含量,降低细胞质膜透性,缓解根系质膜H^ -ATPase活性抑制。以上结果表叫外源海藻糖可能通过增加活性氧清除能力、缓解质膜伤害、维持胞质离子稳态提高植物抗盐性。     

Water content and size of spore components determined by laser diffractometry

For the first time laser diffractometry was used to obtain the refractive index distribution within individual cells in an aqueous environment, allowing accurate determination of their compartmental water content. For bacterial spores the results demonstrated the existence of a dehydrated protoplast surrounded by a less dehydrated integument, thus accounting for the extreme heat resistance of the spores. In contrast, the presence of a dry wall surrounding a water-rich protoplast was shown in fungal spores.     

Effect of hemoglobin A and S on human erythrocyte ghosts

The interaction of erythrocyte ghosts and vesicles with chromatographed hemoglobin (Hb) A and Hb S was studied under various conditions. Although no binding of either Hb A or Hb S to inside-out vesicles was detected, under conditions of physiological ionic strength and pH, several properties of white membrane ghosts were effected by the presence of Hb. Addition of Hb A and Hb S (2 g/dl) to membrane ghosts in 6 mM MgATP, 150 mM NaCl, 10 mM Tris-HCl buffer, pH 7.4, was found to effect the echinocyte-discocyte transition, the extent of endocytosis, the volume, and the sealing of ghosts. Our observations suggest that the structure of membrane ghosts is influenced by cytosol proteins and that the environment of the red cell membrane plays an important role in the definition and the control of the membrane structure and function.     

Detection of Fusarium culmorum in wheat by a surface plasmon resonance-based DNA sensor

A surface plasmon resonance (SPR) sensor based on DNA hybridization has been developed for the detection of Fusarium culmorum, a fungal pathogen of cereals. A 0.57 kbp DNA fragment of F. culmorum was amplified by specific primers and a 25-mer oligonucleotide probe was selected within the sequence of the PCR amplicon. After biotinilation, the probe was immobilized on a streptavidin sensor chip and tested for biospecific interaction with PCR products of F. culmorum. The effect of denaturating agents (formamide and urea) and ionic strength (NaCl) on hybridization efficiency of double-stranded PCR products with the immobilized probe and the specificity of the probe were investigated. The SPR biosensor was successfully used for the detection of F. culmorum in culture material of different strains and in naturally infected wheat samples. Tested on fungal cultures, it showed a good selectivity for F. culmorum against other species of either Fusarium or other fungal genera. A background signal was observed in wheat samples strictly depending on the DNA amount of the testing matrix. Testing 30 ng of durum wheat DNA the detection limit was 0.06 pg of F. culmorum DNA. The developed PCR-SPR assay allowed to detect F. culmorum with sensitivity and specificity higher than gel-electrophoresis analysis.     

The survival of micromycetes and yeasts under the low-temperature plasma generated in electrical discharge

The fungicidal effect of low-temperature plasma generated by positive direct current discharge and its influence on the growth dynamics was evaluated on three micromycete species and yeast in water suspensions. The fungicidal effect was lower than analogous bactericidal effect and differs substantially among various fungal species. Together with the cidal effects, the slower growth of exposed fungal spores was observed.     

Antifungal mechanism of SMAP-29 (1-18) isolated from sheep myeloid mRNA against Trichosporon beigelii

The antifungal activity and mechanism of SMAP-29 (1-18) (SMAP-29), a cathelicidin-derived antimicrobial peptide deduced from N-terminal sequence of sheep myeloid mRNA, were investigated. SMAP-29 displayed a strong antifungal activity against various fungi. To understand the antifungal mechanism(s) of SMAP-29, we examined the interaction of SMAP-29 with the pathogenic fungus Trichosporon beigelii. Confocal microscopy showed that SMAP-29 was localized in the plasma membrane. The antifungal effects of SMAP-29 were further confirmed by using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a plasma membrane probe. Flow cytometric analysis revealed that SMAP-29 acted in an energy-dependent manner. This interaction is also dependent on the ionic environment. Furthermore, SMAP-29 caused significant morphological changes when testing the membrane disrupting activity using liposomes (phosphatidylcholine/cholesterol; 10:1, w/w), as shown by scanning electron microscopy. The results suggest that SMAP-29 may exert its antifungal activity by disrupting the structure of cell membranes, via direct interaction with the lipid bilayers and irregularly disrupted fungal membranes in an energy- and salt-dependent manner.     

Ionic strength-dependent interaction of rat liver casein kinase I with plasma membrane

Casein kinase I binding to rat liver plasma membrane was rapidly released from membrane by increasing the ionic strength above physiological level. The released activities at 250-300 mM NaCl were 3-4-fold higher than those obtained under lower ionic strength below 100 mM NaCl. This reaction occurred nonenzymatically and was reversible. By lowering the ionic strength from 250 mM to 50 mM NaCl by dilution at least 50% of the released enzyme was rebound to plasma membrane. By gel filtration analysis, most of the released enzyme activity under higher NaCl concentration was recovered around the molecular mass of 35,000 which corresponded to that of casein kinase I. This enzyme showed the tendency to aggregate under lower ionic strength (50 mM NaCl), but existed as monomer under higher ionic strength (250 mM NaCl). These results suggest that the release of casein kinase I from plasma membrane and the rebinding to membrane induced by the alteration of ionic strength seem to be an important regulatory mechanism in determining the subcellular distribution of this enzyme.     

Einfluß von Ionenstärke und pH auf die differentielle Dekondensation der Nukleoproteide isolierter Speicheldrüsen-Zellkerne und -Chromosomen von Chironomus thummi

A new method of isolating nuclei and chromosomes of salivary gland cells is described. — The influence of ionic strength and pH of the medium on the state of decondensation of chromosomal bands is studied. In the isolation medium (a modified Ringer solution), all the bands are in a condensed state; as the ionic strength is increased the bands decondense. This reaction of the bands to increasing ionic strength is dependent on the pH which determines: 1) the range of ionic strengths which causes decondensation of the bands; i.e., the lower the pH, the higher the ionic strength is required for decondensation (at pH 7.3, 150–350 mM NaCl, at pH 4.3, 500–800 mM NaCl), and 2) the extent of structural changes caused by increasing ionic strength; that is, at neutral pH the bands become diffuse (“fading”) and at moderate acidic pH (optimum 4.3) the bands unravel to yield pufflike structures (“swelling”). — All ion species tested induce decondensation of bands, but each one is effective differently; specifically, Mg⁺ is more effective than Na⁺ and K⁺, and ClO₄ ^? is more effective than Cl^?. — “Swelling” as induced at pH 4.3 by high ionic strength cannot be reversed by a mere lowering of ionic strength (to 150 mM NaCl) and a subsequent raise of pH (to 7.5); it can be reversed only by an addition of histones. The various histone fractions act differently on the recondensation process. — “Swelling” is correlated with an increase in template activity as evidenced by an increased incorporation of ³H-UTP, measured in the presence of ATP, CTP, GTP and exogeneous RNA polymerase. — The individual bands differ in their sensitivity to an increasing ionic strength. This differential sensitivity expresses itself only if one of the following conditions is met: 1) a moderately acidic pH (optimum 4.3) or 2) the presence of divalent cations at neutral pH. — In a few bands the sensitivity to an increasing ionic strength is dependent on the ionic species (Na⁺, K⁺, Mg⁺⁺ and Ca⁺⁺). — It is attempted to explain the above reactions on the basis of the physico-chemical properties of chromosomes.     

Hydrophobic binding sites on human interferon.

Human interferon binds to a omega-carboxpentyl-agarose column at low ionic strength (0.15 M NaCl) and is still retained when the ionic strength is raised (to 1.0 M NaCl). The binding can be reversed, however, by ethylene glycol, indicating a hydrophobic interaction. The binding of human interferon to omega-aminohexyl-agarose is weak, even at a low ionic strength, and is probably exclusively electrostatic. This disparate binding behavior may be caused by the presence of a positive charge, adjacent to the hydrophobic binding site, on human interferon. The interaction of human interferon with omega-carboxypentyl-agarose is quite selective, inasmuch as the majority of proteins present in interferon preparations pass through the column unretained. Hydrophobic chromatography of human interferon may thus be useful in its purification.     

Physiologically relevant concentrations of NaCl and KCl increase DNA photocleavage by an N-substituted 9-aminomethylanthracene dye

This paper describes the synthesis of a new 9-aminomethylanthracene dye N-substituted with a pyridinylpolyamine side chain (4). The effects of NaCl and KCl on anthracene/DNA interactions were then studied, with the goal of simulating the conditions of high ionic strength that a DNA photosensitizer might encounter in the cell nucleus (~150 mM of NaCl and 260 mM of KCl). As exemplified by methylene blue (5), the expected effect of increasing ionic strength is to decrease DNA binding and photocleavage yields. In contrast, the addition of 150 mM of NaCl in combination with 260 mM of KCl to photocleavage reactions containing micromolar concentrations of 4 triggers the conversion of supercoiled, nicked, and linear forms of pUC19 plasmid into a highly degraded band of DNA fragments (350 nm hν, pH 7.0). Circular dichroism spectra point to a correlation between salt-induced unwinding of the DNA helix and the increase in DNA photocleavage yields. The results of circular dichroism, UV-vis absorption, fluorescence emission, thermal denaturation, and photocleavage inhibition experiments suggest that the combination of salts causes a change in the DNA binding mode of 4 from intercalation to an external interaction. This in turn leads to an increase in the anthracene-sensitized production of DNA-damaging reactive oxygen species.     

An analytical method for the selective retrieval of iminobiotin-derivatized plasma membrane proteins

An analytical method for the selective retrieval of surface plasma membrane proteins which have been covalently "tagged" with the low-molecular-weight ligand 2-iminobiotin has been developed. Retrieval is based upon the specific interaction between the 2-iminobiotin molecule, avidin, antiavidin antibody, and insoluble protein A from Staphylococcus aureus. Conditions for the reaction include moderately basic pH (8.0-9.0) and moderately high ionic strength (300 mM NaCl). The retrieval reaction is insensitive to 4% (w/v) Triton X-100, NP-40, and Lubrols PX and WX and is inhibited by octyl-beta-D-glucopyranoside and Ammonyx-LO. Large numbers of samples can be processed quickly and simultaneously. Immunoprecipitated proteins can be readily released into solution by incubation in the presence of either low pH, biotin, or sodium dodecyl sulfate.     

Spontaneous self-assembly of SC3 hydrophobins into nanorods in aqueous solution

Hydrophobins are small surface active proteins secreted by filamentous fungi. Because of their ability to self-assemble at hydrophilic-hydrophobic interfaces, hydrophobins play a key role in fungal growth and development. In the present work, the organization in aqueous solution of SC3 hydrophobins from the fungus Schizophyllum commune was assessed using Dynamic Light Scattering, Atomic Force Microscopy and fluorescence spectroscopy. These complementary approaches have demonstrated that SC3 hydrophobins are able not only to spontaneously self-assemble at the air-water interface but also in pure water. AFM experiments evidenced that hydrophobins self-assemble in solution into nanorods. Fluorescence assays with thioflavin T allowed establishing that the mechanism governing SC3 hydrophobin self-assembly into nanorods involves β-sheet stacking. SC3 assembly was shown to be strongly influenced by ionic strength and solution pH. The presence of a very low ionic strength significantly favoured the protein self-assembly but a further increase of ions in solution disrupted the protein assembly. It was assessed that solution pH had a significant effect on the SC3 hydrophobins organization. In peculiar, the self-assembly process was considerably reduced at acidic pH. Our findings demonstrate that the self-assembly of SC3 hydrophobins into nanorods of well-defined length can be directly controlled in solution. Such control allows opening the way for the development of new smart self-assembled structures for targeted applications.     

Inter-domain interaction and the structural flexibility of calmodulin in the connecting region of the terminal two domains

The calcium-dependent difference absorption spectrum of scallop calmodulin was measured in the presence of mastoparan. The difference spectrum at 286 nm (delta A286) showed biphasic response to Ca2+ concentration. The first change represents the conformational change around Tyr-138 and the second change may respond to an interaction between N- and C-domain of calmodulin which became apparent in the associated state with mastoparan. Calmodulin-mastoparan complex was eluted from a gel filtration column after free calmodulin in the presence of Ca2+, which indicates a more compact structure of calmodulin-mastoparan complex than of free calmodulin. The biphasic response of delta A286 was also observed with free calmodulin when the ionic strength was as low as 0.02 M NaCl. In the absence of NaCl, the Ca2+ dependence of delta A288 was monophasic, assuming identical affinity of Ca2+ to both domains. Increase in the sensitivity of calmodulin to trypsin was observed with decrease in ionic strength. These results suggest an ionic-strength-dependent decrease in ordered structure of the connecting region. Calmodulin may change shape depending upon the ionic strength by bending at the connecting region. We assumed from the observations that calmodulin in solution may fluctuate between the two extreme shapes of the bent and the dumbbell structure. Target proteins may select and fix the specific bent structure for their activation.     

Characterization of the self-assembly of meso-tetra(4-sulfonatophenyl)porphyrin (H(2)TPPS(4-)) in aqueous solutions

The aggregation of meso-tetra(4-sulfonatophenyl)porphyrin (H(2)TPPS(4-)) in phosphate solutions was investigated as a function of pH, concentration, time, ionic strength, and solution preparation (either from dilution of a freshly prepared 2 mM stock or by direct preparation of μM solution concentrations) using a combination of complementary analytical techniques. UV-vis and fluorescence spectroscopy indicated the formation of staggered, side-by-side (J-type) assemblies. Their size and self-associative behavior were determined using analytical ultracentrifugation and small-angle X-ray scattering. Our results indicate that in neutral and basic solutions of H(2)TPPS(4-), porphyrin dimers and trimers are formed at micromolar concentrations and in the absence of NaCl to screen any ionic interactions. At these low concentrations and pH 4, the protonated H(4)TPPS(2-) species self-assembles, leading to the formation of particularly stable aggregates bearing 25 ± 3 macrocycles. At higher concentrations, these structures further organize or reorganize into tubular, rod-like shapes of various lengths, which were imaged by cryogenic and freeze-fracture transmission electron microscopy. Micron-scale fibrillar aggregates were obtained even at micromolar concentrations at pH 4 when prepared from dilution of a 2 mM stock solution, upon addition of NaCl, or both.     

THE EFFECTS OF SOLUBILIZATION PROCEDURES ON THE RELEASE AND MOLECULAR STATE OF ACETYLCHOLINESTERASE FROM ELECTRIC ORGAN TISSUE

Abstract— A study was made of the effect of various solubilization procedures on the release of AChE from electric organ tissue of the electric eel and on the molecular state of the enzyme. The procedures employed included homogenization in different ionic media or in the presence of detergents, etuymic treatment and chemical modification. Studies were performed on intact electroplax, tissue homogenates and membrane fractions. The apparent AChE activity of intact cells, homogenates and membrane fractions was shown to be governed by diffusion-controlled substrate and hydrogen ion gradients, generated by AChE-catalyscd hydrolysis, leading to a lower substrate concentration and a lower pH in the vicinity of the particulate enzyme.
Treatment of homogenates with NaCl solutions or with NaCl solutions containing the nonionic detergent Triton X-100 causes release of the native'molecular forms of the enzyme (primarily the 18 S species) which aggregate at low ionic strength. For optimal extraction both high ionic strength (e.g. 1 M-NaCl) and the detergent are needed AChE is also solubilized by treatment of tissue homogenates with trypsin, bacterial protease or collagenase. The first two enzymes caused its release as an 11 S non-aggregating form, while collagenase also produces a minor non-aggregating - 16 S component. Treatment of tissue homogenates with maleic anhydride causes release of AChE as a non-aggregating 18 S species. On the basis of the solubilization experiments it is concluded that the interaction of AChE with the excitable membrane is primarily electrostatic. The possible orientation of the enzyme within the synaptic gap is discussed.