Polyamines and nucleic acids during development of the chick embryo

1. A higher concentration of polyamines (spermine, spermidine, putrescine and cadaverine) during development of the chick embryo was observed between the fifth and tenth day of incubation; the concentrations of nucleic acids showed a parallel increase. 2. When spermine (5mumoles) was injected into the yolk sac of embryos at the tenth day of incubation, a high amine-oxidase activity was noted and the spermine and spermidine concentrations were decreased; also, there was a remarkable decrease in RNA and DNA concentrations and a parallel increase in that of total free nucleotides. 3. On the other hand, when iproniazid (16mumoles) was injected there was an inhibition of amine-oxidase activity and a similar increase in the concentrations of spermine and spermidine and of nucleic acids, whereas that of total free nucleotides decreased. 4. Another group of embryos injected with spermine and iproniazid together showed a remarkable increase in spermine and spermidine concentrations and a parallel increase in those of RNA and DNA, and a decrease in that of total free nucleotides.     

Properties of L-glutamate decarboxylase from brains of adult and newborn mice.

Abstract— l -Glutamate 1-carboxy-lyase (EC 4.1.1.15) (GAD) and 4-aminobutyrate-2-oxo-glutarate aminotransferase (EC 2.6.1.19) (GABA-T) have been purified from mouse brain (Wu et al. 1973; Schousboe et al., 1973) and their properties have been extensively studied (Wu & Roberts , 1974; Schousboe et al., 1974). The above enzymes were prepared from a water lysate of crude mitochondrial fraction, which accounted for only 25–30% of total GAD or GABA-T activities in brain. A procedure has been developed which liberates approx 85% of total GAD and GABA-T activities into supernatant. Two distinct, well-separated peaks with GAD activity and a single peak with GABA-T activity were observed when a concentrated extract from brain of adult or newborn mice was chromatographed on Sephadex G-200 or Bio-Gel A–1.5 m. The first peak appeared in the void volume and is. therefore. an entity of high molecular weight. The second peak gave elution characteristics which were identical to those of the enzyme that had been purified previously (mol wt = 85,000). These two GAD peaks were also clearly separated on polyacrylamide gel electrophoresis. The GAD activities in the two peaks showed similar pH profiles (optimum, 6.5). K_m values (1–2 mM), immunodiffusion patterns and inhibitions by anti-GAD IgG prepared against GAD purified from synaptosome-containing crude mitochondrial fraction (60–80%). The physiological implications of high molecular weight and low molecular weight forms of GAD are discussed.     

Polyamines and nucleic acid metabolism in chick embryo. Incorporation of labelled precursors into nucleic acids of subcellular fractions and polyribosomal patterns

1. An increase in polyamine concentration, caused by inhibiting the amine oxidase activities with iproniazid, increased the incorporation of [³H]orotic acid into chick-embryo RNA and DNA. On the other hand, a decrease in polyamine concentration, obtained by causing an increase in amine oxidase activities, decreased [³H]orotic acid incorporation into nucleic acids. This was particularly evident for nuclear DNA and ribosomal RNA. 2. Polyribosomal patterns obtained by sucrose-density-gradient centrifugation showed highest radioactivity in the regions of 259s and 280s aggregates in those embryos in which the polyamine contents were enhanced, whereas a decrease in the radioactivity was observed when the polyamine concentrations were decreased. 3. The activity of DNA-dependent RNA polymerase, assayed in the same experimental conditions, also varied in the same fashion with changes in polyamine concentration.     

Polyamines bound to nucleic acids during dormancy and activation of tuber cells of Helianthus tuberosus

Tuber tissue of Helianthus tuberosus L. (cv. OB1) contains a low amount of polyamines during dormancy but they are rapidly synthesized when tuber cells are activated in a growth medium and enter a new cell cycle. It was assumed that one of the reasons for this synthesis is that polyamines are necessary for the active conformation and correct functioning of nucleic acids. Complexes were found between spermine, spermidine and putrescine and rRNA, tRNA and an RNA fraction which contains poly(A) RNA and proteins. The amount of RNA-bound polyamines in the parenchyma cells of dormant tubers is dependent on the stage of dormancy and clearly increases (especially putrescine) when cells are activated. There are both tightly-bound and non-tightlybound polyamines. The significance of these bound polyamines is discussed in relation to their stabilizing role on nucleic acids.     

Interaction of nucleic acids. VI. Interaction of purine with nucleic acids

The interaction of purine with DNA, tRNA, poly A, poly C, and poly A. poly U complex was investigated. In the presence of purine, the nucleic acids in coil form (such as denatured DNA, poly A and poly C in neutral solutions, or tRNA) have lower optical rotations. In addition, hydrodynamic studies indicate that in purine solutions the denatured DNA has a higher viscosity and a decreased sedimentation coefficient. These findings indicate that through interaction with purine, the bases along the poly-nucleotide chain are unstacked and are separated farther from each other, resulting in increased assymmetry (and possibly volume) of the whole polymer. Thus, the de-naturation effect of purine reported previously can be explained by this preferential interaction of purine with the bases of nucleic acids in coil form through a hydrophobic-costacking mechanism. Results from studies on optical rotation and helix-coil transition show that the interaction of purine is greater with poly A than with poly C. The influence of temperature, Mg⁺⁺ concentration, ionic strength, and purine concentration on the effect of purine on nucleic acid conformation has also been investigated. In all these situations the unraveling of nucleic acid conformation occurs at much lower temperatures (20–40°C lower) in the presence of purine (0.2–0.6M).     

Quadruplex structures in nucleic acids.

DNA oligonucleotides that have repetitive tracts of guanine bases can form G-quadruplex structures that display an amazing polymorphism. Structures of several new G-quadruplexes have been solved recently that greatly expand the known structural motifs observed in nucleic acid quadruplexes. Base triads, base hexads, and quartets that contain cytosine have recently been identified stacked over the familiar G-quartets. The current status of the diverse array of structural features in quadruplexes is described and used to provide insight into the polymorphism and folding pathways. This review also summarizes recent progress in the techniques used to probe the structures of G-quadruplexes and discusses the role of ion binding in quadruplex formation. Several of the quadruplex structures featured in this review can be accessed in the online version of this review as CHIME representations.     

Polyamines and nucleic acids during the first cell cycle of Helianthus tuberosus tissue after the dormancy break

Polyamines (spermine, spermidine, and putrescine) and nucleic acids were studied during the first cell cycle after the break of dormancy of tuber slices of Helianthus tuberosus L., cv. OB1. Immediately after the break of dormancy, a marked decrease in stored arginine and glutamine and a corresponding increase of polyamines were observed. This first synthesis of polyamines were observed. This firs synthesis of polyamines occurred very early during the G₁ phase, concomitant to the synthesis of RNAs. A RNA, probably messenger-like RNA, was synthesized very actively only during the first hours of activation in the culture medium plus 2,4-dichlorophenoxyacetic acid, or in water. At the onset of the S phase, after 12h of activation, an incorporation of [³H] thymidine was also detected. A second putrescine synthesis and polyamine accumulation began during the progression of the S phase. During the progression of mitosis, there was a decrease of polyamine synthesis and accumulation.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - GA₇ Gibberellin A₇ - MAK methylated albumin column     

Larval and adult brains

Apical organs are a well-known structure in almost all ciliated eumetazoan larvae, although their function is poorly known. A review of the literature indicates that this small ganglion is the "brain" of the early larva, and it seems probable that it represents the brain of the ancestral, holopelagic ancestor of all eumetazoans, the gastraea. This early brain is lost before or at metamorphosis in all groups. Protostomes (excluding phoronids and brachiopods) appear to have brains of dual origin. Their larvae develop a pair of cephalic ganglia at the episphere lateral to the apical organ, and these two ganglia become an important part of the adult brain. The episphere and the cerebral ganglia show Otx expression, whereas Hox gene expression has not been seen in this part of the brain. A ventral nervous system develops around the blastopore, which becomes divided into mouth and anus by fusion of the lateral blastopore lips. The circumblastoporal nerve ring becomes differentiated into a nerve ring around the mouth, becoming part of the adult brain, a pair of ventral nerve cords, in some cases differentiated into a chain of ganglia, and a ring around the anus. This part of the nervous system appears to be homologous with the oral nerve ring of cnidarians. This interpretation is supported by the expression of Hox genes around the cnidarian mouth and in the ventral nervous system of the protostomes. The development of phoronids, brachiopods, echinoderms, and enteropneusts does not lead to the formation of an episphere or to differentiation of cerebral ganglia. In general, a well-defined brain is lacking, and Hox genes are generally not expressed in the larval organs, although this has not been well studied.     

Spin labeled nucleic acids.

Homopolyribonucleotides and E. coli DNA wer spin labeled with an iodoacetamide-nitroxide compound. The extent of labeling is highly dependent upon the nature of the base and the secondary structure of the nucleic acid. This spin label-polymer linkage is unstable at high temperatures and in phosphate buffers. In order to determine the effect of changes in the environment of nucleic acids on the esr signals of their attached spin labels, the polynucleotides were subjected to temperature and viscosity perturbations. An increase in temperature (T) affects a linear decrease in the anisotropy factor of the esr signal. The log tau (tau = correlation time) versus (1/T) profile is linear with a positive slope when the spin label is attached to single stranded polynucleotides but exhibits discontinuities at certain critical temperatures when attached to the duplexes poly (As-U) and poly (I-Cs). These critical temperatures are lower than the optical Tm. Logarithmic increase in viscosity was found to produce a linear increase in tau in aqueous sucrose solutions.     

Iodination of herpesvirus nucleic acids.

A simple method is described for the iodination of herpes simplex virus (HSV) DNA. The procedure involved synthesis of 125-I-labeled 5-iodo-dCTP which was subsequently used as a precursor for the in vitro repair synthesis of HSV DNA. Synthesis of 5-iodo-dCTP and purification from oxidation and reduction reagents, buffer salts, unreacted dCTP and Na125-I was accomplished in a single chromatographic step. It was possible to prepare 125-I-labeled HSV DNA in vitro with specific activities exceeding 10-8 counts/min/mu-g. The DNA prepared by this method reassociated with DNA extracted from HSV-infected HEp-2 cells but not with HEp-2 cell DNA. Iodinated HSV DNA was susceptible to S-1-endonuclease digestion once denatured but was resistant to digestion in the native form. This method was used to synthesize 125-I-labeled ribo-CTP (5-iodo-CTP) which was used to prepare cytomegalovirus-specific complementary RNA. The method should be of value in the preparation of viral probes and for use in autoradiography of viral nucleic acids.     

Trinitrophenylation of nucleic acids and their constituents.

1. Under relatively mild conditions, nucleic acids and their constituents were trinitrophenylated with 2,4,6-trinitrobenzenesulfonate (TNBS) in aqueous solution (pH 8-11), yielding reddish-orange trinitrophenyl (TNP) derivatives. Guanine residues were trinitrophenylated on the base residues at the 2-amino group (N2-TNP derivatives), and in addition, 2'- and 3'-hydroxyl groups of the ribose moieties of nucleosides or nucleotides were trinitrophenylated to form Meisenheimer complexes. 2. The preparation of TNP derivatives (N2-TNP-guanine, -guanosine, N2, O-bis-TNP-guanosine, O-TNP-guanosine, -adenosine, -cytidine , and -uridine), their rates of formation, absorption spectra (UV, visible, and infrared), molar extinction coefficients, Rf value, electrophoretic mobilities, and stability in acid or alkaline solution, are presented. 3. Trinitrophenylation of several kinds of nucleic acid was investigated. Calf thymus DNA and yeast transfer RNA showed a resistance to trinitrophenylation compared to guanosine 3'(2')-phosphate, yeast RNA or denatured calf thymus DNA. TNP-RNA showed resistance to the action of ribonucleases T1 and T2 [EC 3.1.4.8 and 3.1.4.23]. 4. Trinitrophenylation reactions using 2,4,6-trinitrochlorobenzene and 2,4,6-trinitrofluorobenzene were compared with that using TNBS as regards specificity and reaction rate.     

Isolation and characterization of adult rat hearts cells.

Adult rat heart muscle cells were isolated after simultaneous perfusion of multiple (two to eight) hearts with buffered salt solutions containing collagenase and hyaluronidase. Yields (35 to 50% of ventricular weight with approximately 70% viability) are quantitatively suitable for metabolic studies. Viability has been determined by the ability of intact cells to exclude trypan blue and the inability of intact cells to oxidize exogenous succinate. Micrographs show that the fine structure of the isolated cells is well ordered. Cell concentrations of glycogen, glucose 6-phosphate, citrate, and various enzymes were similar to those of intact heart. ATP and creatine phosphate concentrations were lower than in whole hearts. Adenosine 3′,5′-monophosphate concentrations were somewhat elevated. Deoxyribonucleic acid was lower than in whole tissue. The isolated cells retain certain metabolic control mechanisms. The uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, increased oxygen consumption severalfold, whereas exogenous ADP had no effect on respiration. Under anaerobic conditions the rates of glucose utilization and lactate production were faster than in the presence of oxygen, indicating retention of the Pasteur effect. The addition of glucose and insulin caused a decrease in oxygen uptake or the Crabtree effect. Exogenously added pyruvate decreased glycolytic flux and produced a pronounced increase in intracellular citrate and glucose 6-phosphate. Isoproterenol stimulated adenylate cyclase activity of the isolated cells at the same concentrations effective with intact heart preparations. Isoproterenol and glucagon caused the activation of phosphorylase. The cells deteriorated as a function of incubation time, as indicated by a decrease in ATP content and a loss of lactate dehydrogenase into the medium. Cell deterioration was greatly accelerated by Ca²⁺ at concentrations greater than 10^?5m.     

Conformational classification of functional nucleic acids.

The kink parameters would provide the tolerant aspect for irregular helical structure of nucleic acid. Using these kink parameters, the classification of conformation space was carried for the functional nucleic acid molecules. The kink parameters could afford us the simple structural aspects about the constructive parts of functional molecules. Local elastic kink phenomena can be classified by rod like models with the combination of kink parameters. The constructive parts, such as the stable tetra nucleotides loop, U-turn conformation and adenosine platform, were selected and the statistical analyses were carried on the parameters calculated by program BIOCON.