Melting profiles for lymphocyte chromatin of healthy subjects and Down's disease patients obtained by the technic of cellular DNA absorption in ultraviolet light

Integral values of optical density (E260) were obtained for lymphocyte nuclei from normal people and those suffering from Down's syndrome during the melting of cells in media of varying ionic strength (0.15 M NaCl - control; 0.015, 0.0015 and 0.00015 M NaCl - experimental). The differences in E260 were obtained only during the melting of cells in 0.15 M NaCl. In lymphocytes from normal people, the distinctly reproducible hyperchromic effect was detected from 78 degrees (up to 35--40%), reaching the plateau by 96 degrees C (P less than 0.01). In identical conditions, the hyperchronic effect on aneuploid cells was revealed only after 98 degrees C (P less than 0.01). The data presented confirm the authors' concept of the greater condensation of aneuploidic genome.     

Opposing effects of NaCl on reversibility and thermal stability of halophilic beta-lactamase from a moderate halophile, Chromohalobacter sp. 560

Beta-lactamase from a moderately halophilic organism is expected to show salt-dependent stability. Here we examined the temperature-dependence of stability at different salt concentrations using circular dichroism (CD) and enzyme activity. NaCl showed opposing effects on melting temperature and reversibility of the thermal melting. Increasing NaCl concentration greatly increased the melting temperature from, e.g., 41 degrees C in the absence of NaCl to 61 degrees C in 3 M NaCl. Conversely, reversibility decreased from 92% to 0% in the corresponding NaCl solutions. When beta-lactamase was heated at different temperatures and NaCl concentrations, the activity recovery followed the reversibility, not the melting temperature. Heating beta-lactamase at 63 degrees C, slightly above the onset temperature of melting in 2 M NaCl and far above the melting in 0.2 M NaCl, showed a much greater recovery of activity in 0.2 M NaCl than in 2 M NaCl, again consistent with the reversibility of melting.     

Phosphorylation and DNA binding of nuclear rat liver proteins soluble at low ionic strength.

Proteins were extracted from isolated rat liver nuclei with 0.15 M NaCl and 0.35 M NaCl at pH 8.0. The number of phosphoproteins in these extracts was determined by labeling with 32P and autoradiography after two-dimensional gel electrophoresis. Two proteins, B22p and B24p, contained small amounts of 32P and sedimented with the 30S nuclear informofer particle. With the exception of two phosphoproteins, CB and CN', all of the phosphoproteins found in the 0.35 M NaCl extract. Approximately 20% of the 0.15 M NaCl soluble proteins bound to rat liver DNA in 0.05 M KCl-0.05 M Tris-HCl (pH 8). Of these proteins, 1-2% bound to DNA in 0.15 M KCl and were eluted with 2 M KCl. This DNA bound fraction which contained both phosphorylated and nonphosphorylated proteins was similar in both the 0.15 and 0.35 M NaCl extracts. However, two major proteins (C13 and C14) and three minor proteins (C15, C25, Cg') were present only in the 0.15 M NaCl extract. The results of the present study show that there are marked similarities in the two-dimensional gel electrophoretic, phosphorylation, and DNA binding properties of rat liver nuclear proteins soluble in either 0.15 or 0.35 M NaCl.     

Destabilization of secondary structure in 16S ribosomal RNA by dimethylation of two adjacent adenosines.

Fragments comprising the 49 nucleotides from the 3'-end have been purified from 16S ribosomal RNA of wild-type Escherichia coli and from a kasugamycin-resistant mutant that specifically lacks dimethylation of two adjacent adenines near the 3'-terminus. These fragments, obtained after treatment of ribosomes in vitro with the bacteriocin cloacin DF13, were used to study the effect of the methyl groups on the temperature dependent unfolding of double-stranded regions. Both fragments contain at least 3 independent melting transitions, of which the one with the highest Tm corresponds with the unfolding of a nine-basepair long central hairpin. Dimethylation of the adenines in the loop of this hairpin lowers the melting temperature (Tm) by approximately 2 degrees C at 0.2 M NaCl and by about 5 degrees C at 0.15 M NaCl. It is suggested that m6(2)Am6(2)A is more antagonistic to loop formation that ApA and that the function of the methyl groups is to help to destabilize the 3'-terminal hairpin in 16S rRNA in order to facilitate intermolecular interactions.     

B--Z conformational transition in d(CGCGCGTTAATT), d(CGCGCGTTAA) and d(CGCGCGTT)

Conformational studies on three DNA-oligomers (d(CGCGCGTTAATT), d(CGCGTTAA) and d(CGCGCGTT) in solution by circular dichroism spectroscopy are reported. In low salt solution, all three DNA oligomers exhibit a characteristic B-conformation. However, under the influence of high salt concentration i.e. 5M NaCl, the octamer d(CGCGCGTT) exhibits 'A' conformation whereas the decamer and dodecamer retain B-conformation. On addition of millimolar amount of NiCl2 to the 5M NaCl, solution of oligodeoxynucleotides a B-Z transition is observed in octamer, decamer and dodecamer. However, NiCl2 titrations show that mid point of transition for dodecamer is at 2.25 mM, for decamer is at 13 mM NiCl2 and for octamer is 17 mM at NiCl2. In 60% alcohol all three oligonucleotides remain in the B-conformation. The melting temperatures of oligonucleotides at various salt concentration are also reported. Thermodynamic parameters calculated by melting profile using a two state model show that dodecamer and decamer are most stable in their 5M NaCl, B-form. However, octamer is more stable in its Z form than that of its 'A' form.     

Number of water molecules coupled to the transport of sodium, potassium and hydrogen ions via gramicidin, nonactin or valinomycin.

The number of water molecules (n) coupled to the transport of cations across lipid membranes was determined in two different wats: directly from the electro-osmotic volume flux per ion, and by the use of Onsager's relation, from the open circuit streaming potential produced by an osmotic pressure difference. The results of the two approaches were in general agreement. Monoolein membranes were formed on the ends of polyethylene or Teflon tubing connected to a microliter syringe and the volume change necessary to keep the membrane at a fixed position was measured. It was necessary to make corrections for unstirred layer effects. The results for gramicidin were: n approximately 12 for 0.15 M KCl and NaCl, n approximately 6 for 3.0 M KCl and NaCl, and n approximately 0 for 0.01 M HCl. For nonactin, n approximately 4 for both 0.15 and 3.0 M KCl and NaCl. Valinomycin (for 0.15 M KCl) behaved like nonactin. It is shown that for a channel mechanism, in general, n is less than or equal to the number of water molecules in a channel that does not contain any cations. Thus, the n of 12 for the 0.15 M salts implies that the gramicidin channel can hold at least 12 water molecules. This places an important constraint on models of the channel structure. The n of 0 for HCl is consistent with a process in which protons jump along a continuous row of water molecules. The decrease of n with the 3.0 M salts may indicate that the channel becomes multiply occupied at high salt concentrations. The n of 4 for nonactin and valinomycin means that at least four water molecules are associated with the carrier . cation complex, probably in the interstices between the complex and the disordered lipid.     

Tachykinin NK3 receptor contribution to systemic release of vasopressin and oxytocin in response to osmotic and hypotensive challenge

Activation of the neurokinin 3 receptor (NK3R) by a receptor agonist, hypotension, and hyperosmolarity results in the internalization of NK3R expressed by magnocellular neurons and the release of vasopressin (VP) and oxytocin (OT) into the circulation. The contribution of NK3R activation to the release of VP and OT in response to hyperosmolarity and hypotension was evaluated by measuring the release of both hormones following pretreatment with a selective NK3R antagonist, SB-222200. Freely behaving male rats were given an intraventricular injection of either 0.15 M NaCl or 250, 500, or 1,000 pmol SB-222200, and then were administered an intravenous infusion of 2 M NaCl or 0.15 M NaCl (experiment 1), or a bolus intra injection of 0.15 M NaCl or hydralazine (HDZ), a hypotension-inducing drug (experiment 2). Blood samples were taken from indwelling arterial catheters at various time points for 1-2 h, both before and after treatments. Plasma VP and OT levels were determined by ELISA. Blockade of NK3R did not affect the baseline levels of either hormone. In contrast, pretreatment with SB-222200 significantly reduced ( approximately 60%) or abolished the release of VP and OT, respectively, to 2 M NaCl infusion. HDZ-induced VP and OT release was eliminated by pretreatment with 500 pmol SB-222200. Therefore, NK3R activation contributes significantly to the systemic release of both VP and OT in response to osmotic and hypotensive challenges.     

Presystemic influences on thirst, salt appetite, and vasopressin secretion in the hypovolemic rat

The present studies investigated the influence of presystemic signals on the control of thirst, salt appetite, and vasopressin (VP) secretion in rats during nonhypotensive hypovolemia. Rats were injected with 30% polyethylene glycol (PEG) solution, deprived of food and water overnight, and then allowed to drink water, 0.15 M NaCl, or 0.30 M NaCl. The PEG treatment, which produced 30-40% plasma volume deficits, elicited rapid intakes in an initial bout of drinking, but rats consumed much more 0.15 M NaCl than water or 0.30 M NaCl. In considering why drinking stopped sooner when water or concentrated saline was ingested, it seemed relevant that little or no change in systemic plasma Na(+) concentration was observed during the initial bouts and that the partial repair of hypovolemia was comparable, regardless of which fluid was consumed. In rats that drank 0.15 M NaCl, gastric emptying was fastest and the combined volume of ingested fluid in the stomach and small intestine was largest. These and other observations are consistent with the hypothesis that fluid ingestion by hypovolemic rats is inhibited by distension of the stomach and proximal small intestine and that movement of dilute or concentrated fluid into the small intestine provides another presystemic signal that inhibits thirst or salt appetite, respectively. On the other hand, an early effect of water or saline consumption on VP secretion in PEG-treated rats was not observed, in contrast to recent findings in dehydrated rats. Thus the controls of fluid ingestion and VP secretion are similar but not identical during hypovolemia.     

Regions necessary for pH-dependent assembly of gizzard myosin rod

Aggregated and disaggregated forms of gizzard myosin rod and its fragments in various concentrations of NaCl (0-0.30 M) at various pH (7.4-8.6) were distinguished from each other by their permeability through a Sepharose 4B column. The rod existed in three forms, namely: large aggregates impermeable to the column, small aggregates eluted at the void volume of the column and a disaggregated monomer which penetrated the column. The relative proportions of the three forms varied depending on the salt concentration and pH. The monomeric rod was detected in NaCl solutions above 0.20 M and its relative proportion at 0.25 M NaCl was larger than those of the small and large aggregates. The small aggregates of the rod were predominant at below 0.05 M NaCl and, upon decrease in pH from 8.6 to 7.4, these small aggregates in NaCl solutions between 0.10 M and 0.15 M were replaced by the large aggregates. Light meromyosin, which corresponded to the C-terminal two-thirds of the rod, existed exclusively as large aggregates in NaCl solutions below 0.15 M; increase of NaCl concentration to above 0.20 M resulted in the formation of its monomer, instead of the large aggregates. In contrast to the rod, no small aggregated form of the light meromyosin was detected. Truncated light meromyosin which had lost a small segment from either the C-terminal or N-terminal of light meromyosin was eluted only as a monomer in any NaCl concentration at any pH. It may be deduced from the above results that a small segment in the light meromyosin is requisite for the assembly of both rod and light meromyosin in the NaCl solutions below 0.15 M and that the relative proportion of small and large aggregates of the rod is determined in a pH-dependent manner by the subfragment 2 segment, the N-terminal third of the rod.     

H3 Cys-110 is in close proximity to the C-terminal regions of H2B and H4 in a nucleosome core with an altered internal arrangement of histones

A particle obtained by nuclease digestion of nucleohistone complexes prepared by direct mixing of histones with DNA in 0.15 M NaCl was indistinguishable by composition and physical properties from nucleosome cores prepared under the same conditions from nucleohistone preannealed in 0.6 M NaCl. We show here that different photo-cross-links form when these particles are prepared from H3 labeled with photoaffinity reagents on the unique histone H3 cysteine. H3-H3 histone dimers were dominant when the particles were prepared by dilution of the nucleohistone from 0.6 M NaCl while H3-H2B and H3-H4 histone dimers were prominent if the nucleohistone complex was prepared directly in 0.15 M NaCl. Peptide mapping of the novel H3-H4 and H3-H2B dimers showed that Cys-110 of histone H3 is cross-linked to the 18 amino acid C-terminal end of H4 or to the 66 amino acid C-terminal half of H2B.     

Direct measurement of osmotic pressure of glycosaminoglycan solutions by membrane osmometry at room temperature

Articular cartilage is a hydrated soft tissue composed of negatively charged proteoglycans fixed within a collagen matrix. This charge gradient causes the tissue to imbibe water and swell, creating a net osmotic pressure that enhances the tissue's ability to bear load. In this study we designed and utilized an apparatus for directly measuring the osmotic pressure of chondroitin sulfate, the primary glycosaminoglycan found in articular cartilage, in solution with varying bathing ionic strength (0.015 M, 0.15 M, 0.5 M, 1 M, and 2 M NaCl) at room temperature. The osmotic pressure (pi) was found to increase nonlinearly with increasing chondroitin sulfate concentration and decreasing NaCl ionic bath environment. Above 1 M NaCl, pi changes negligibly with further increases in salt concentration, suggesting that Donnan osmotic pressure is negligible above this threshold, and the resulting pressure is attributed to configurational entropy. Results of the current study were also used to estimate the contribution of osmotic pressure to the stiffness of cartilage based on theoretical and experimental considerations. Our findings indicate that the osmotic pressure resulting from configurational entropy is much smaller in cartilage (based on an earlier study on bovine articular cartilage) than in free solution. The rate of change of osmotic pressure with compressive strain is found to contribute approximately one-third of the compressive modulus (H(A)(eff)) of cartilage (Pi approximately H(A)(eff)/3), with the balance contributed by the intrinsic structural modulus of the solid matrix (i.e., H(A) approximately 2H(A)(eff)/3). A strong dependence of this intrinsic modulus on salt concentration was found; therefore, it appears that proteoglycans contribute structurally to the magnitude of H(A), in a manner independent of osmotic pressure.     

Immunofluorescent localization of lysine-rich histones in isolated nuclei from adult and embryonic chicken erythrocytes

Isolated nuclei from adult chicken erythrocytes were stained by indirect immunofluorescence for histones H5 and H1. Nuclei in 0.15 M NaCl stained for H5 showed internuclear variations in intensity of fluorescence from bright to dim. Most individual nuclei were homogeneously stained although some showed a bright rim around a dimmer interior. Treatment of nuclei with Tween 80 in 0.15 or 0.03 M NaCl also gave internuclear variation in intensity. Adult nuclei stained for H1 (in 0.15 or 0.03 M NaCl) showed little internuclear variation; most nuclei stained brightly with a brighter rim. Simultaneous staining of H5 and H1 in the same nuclei confirmed the variable fluorescence of H5 and consistent fluorescence of H1. Most nuclei showed the presence of both histones. Nuclei from embryonic blood cells also showed considerable internuclear variation of H5 fluorescence and less variation with H1 staining. For both histones the proportion of brightly staining nuclei increased with embryonic development. Difficulties in interpreting quantitative variations in immunofluorescence are discussed.     

Subcellular distribution of DNA-binding proteins from cultured hamster fibroblasts

The distribution between nuclei and cytoplasm of DNA-binding proteins from growing NIL cells was studied. To obtain the subcellular fractions, cell monolayers or cells previously detached from the culture dish were treated with the non-ionic detergent Nonidet P-40. Proteins with affinity for DNA were isolated from nuclear or cytoplasmic fractions by chromatography on DNA-cellulose columns and were further analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results show that P8, one of the major components in the 0.15 M NaCl-eluted proteins, is found predominantly in the cytoplasmic fractions, whereas P6, the other main protein peak in this eluate, is more prominent in the nuclear fraction. Among the other proteins eluted at 0.15 M NaCl from the DNA-cellulose column, P5 and P5′ are detected in both nuclear and cytoplasmic fractions. All the other proteins in the 0.15 M NaCl eluate are present almost exclusively in the cytoplasmic fraction. On the other hand, most of the proteins with higher affinity for DNA, eluted from the column at 2 M NaCl, are present in the nuclear fraction, although they are also detected in the cytoplasm in amounts similar to those observed in the nuclei.     

Lactate and malate dehydrogenase binding to the microsomal fraction from chicken liver

Chicken liver microsomal fractions show lactate and malate dehydrogenase activities which behave differently with respect to successive extractions by sonication in 0.15 M NaCl, 0.2% Triton X-100 and 0.15 M NaCl, respectively. The Triton X-100-treated pellet did not show malate dehydrogenase activity but exhibited a 10-fold increase in lactate dehydrogenase activity with respect to the sonicated pellet. Total extracted lactate and malate dehydrogenase activities were, respectively, 7.5 and 1.7 times higher than that in the initial pellet. Different isoenzyme compositions were observed for cytosoluble and microsomal extracted lactate and malate dehydrogenases. When the ionic strength (0-500 mM) or the pH values (6.1-8.7) of the media were increased, an efficient release of lactate dehydrogenase was found at NaCl 30-70 mM and pH 6.6-7.3. Malate dehydrogenase solubilization under the same conditions was very small, even at NaCl 500 mM, but it attained a maximum in the 7.3-8.7 pH range. Cytosoluble lactate dehydrogenase bound in vitro to 0.15 M NaCl-treated (M2) and sonicated (M3) microsomal fractions but not to the crude microsomal fraction (M1). Particle saturation by lactate dehydrogenase occurred with M2 and M3, which contained binding sites with different affinities. Cytosoluble malate dehydrogenase did not bind to M1, M2 and M3 fractions, however, a little binding was found when purified basic malate dehydrogenase was incubated with M2 or M3 fractions.     

Discrimination of assembled and disassembled forms of gizzard myosin by papain

Chicken gizzard myosin in 0.15 M or 0.5 M NaCl was cleaved at two sites of heavy chain with 2-10 micrograms/ml papain. MgATP inhibited these cleavages of myosin in 0.15 M NaCl but not in 0.5 M NaCl. The protective effect of ATP was observed at concentrations as low as 10 microM and increased in proportion to ATP concentration to a maximum at 1 mM. ADP was as effective as ATP, while adenosine 5'-[beta, gamma-imido]triphosphate, an unhydrolyzable ATP analogue, was less effective than ATP or ADP. AMP had no protective effect on the digestion of myosin and GTP inhibited slightly the digestion. When the papain-insensitive myosin in 0.15 M NaCl and 2.5 mM MgATP was phosphorylated by Ca2+/calmodulin-dependent myosin light-chain kinase, the myosin restored the vulnerability to papain. However, the two papain-susceptible forms, nonphosphorylated form in the absence of MgATP and phosphorylated form in the presence of MgATP, yielded very similar but distinct proteolytic fragments upon the digestion. When the extent of myosin assembly was estimated by the turbidimetry of myosin suspension in 0.15 M NaCl, nonphosphorylated myosin in the absence and presence of MgATP was assembled and disassembled, respectively, and phosphorylated myosin in the presence of MgATP was assembled. These results suggest that, at physiological ionic strength, papain as a probe distinguishes disassembled myosin and assembled myosin as papain-insensitive and papain-sensitive forms, respectively.     

Histones in the first cleavage cycle of fertilised sea urchin eggs

Nuclei were isolated from Echinus eggs through the first cleavage cycle by modification of existing techniques. When these nuclei were extracted with 2 M NaCl and the supernatant diluted to 0.15 M, large amounts of non-histone proteins remained in solution. The precipitated nucleoprotein contained expected amounts of DNA and a protein analogous to mammalian histone H1. Extrachromosomal histone H1 was eliminated by the modified isolation procedure. Amounts of nuclear proteins soluble in 0.15 M NaCl reached a peak in G2. Histones and non-histone proteins were phosphorylated postfertilization, in early prophase and in telophase.     

Potentiality of artificial sea water salts for the production of carrageenase by a marine Cytophaga sp

Production of an extracellular enzyme complex (carrageenase) was studied by examining cell-free fluids from cultures of a marine Cytophaga, 1k-C783, growing on different media. Among artificial sea water salts, only NaCl and MgCl2 were utilized by the organism to produce carrageenase. The minimal concentrations of suitable combinations of NaCl and MgCl2 were found to be 0.05 M NaCl plus 0.25 M MgCl2, and 0.15 M NaCl plus 0.15 M MgCl2. KCl and CaCl2 did not have any role in carrageenase production in ZoBell 2216 E broth medium. Carrageenase was synthesized continuously within the resting cells and was released from the cells as well as in the growing cells, when nutrient had been supplied.     

Compact Escherichia coli nucleoids in a highly supercoiled conformation.

Escherichia coli nucleoids were visualized in a compact, highly supercoiled conformation at 1 M NaCl. After the salt concentration was lowered to 0.15 M NaCl, the nucleoids uncoiled in beaded fibers in which RNA-mediated structural domains could be distinguished.     

Calorimetric study of the complexes between polyuridylic acid and adenylic nucleotides.

Soluble complexes of poly (U) and adenylic nucleotides in NaCl solutions were studied by scanning microcalorimetry. The melting enthalpies, delta Hm, of poly (U) complexes with adenosine, 2',3' -cAMP, 2'(3')-AMP, 5-AMP, ADP, ATP in 1 M NaCl are 50.5; 45.0; 42.9; 28.6; 26.1 and 25.6 kJ/mole triplets, respectively. Delta Hm is independent of the complex melting temperature, Tm. The calorimetric enthalpies are considerably lower than the apparent delta Hv.H. obtained from Tm dependence on free monomer concentration. The enthalpy of complex formation in 1 M NaCl depends neither ob the number nor on the degree of ionization of the phosphate groups but is essentially determined by their 5' - or 2'(3')-position. In contrast to 2'(3')- AMP. 2 poly (U), delta Hm of 5'AMP. 2 poly (U) increases considerably at lowering Na+ concentration. The enthalpy of poly (U) double helix melting in 1 M NaCl is 8.8 kJ/mole pairs which is 2.5 times lower than that in MgCl2 solutions.     

Identification of a silver binding protein associated with the cytological silver staining of actively transcribing nucleolar regions.

Nucleoli isolated from Novikoff hepatoma cells were stained with AgNO3 to demonstrate the typical staining of active ribosomal cistrons. Pre-treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 2.0 M NaCl did not interfere with silver staining. Treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 0.15 M NaCl did, however, eliminate silver binding. Serial extraction of nucleoli with 2.0 M NaCl buffer followed by 0.15 M NaCl buffer also abolished silver staining. Analysis of the supernatant fraction of these extracts by polyacrylamide gel electrophoresis indicates that, although more than one nucleolar protein can bind silver, only one protein is associated with the staining of active ribosomal cistrons.