Methidiumpropyl-EDTA-iron(II) cleavage of ribosomal DNA chromatin from Dictyostelium discoideum.

We have used methidiumpropyl-EDTA-iron(II) [MPE.Fe(II)] in parallel with micrococcal nuclease to investigate the chromatin structure of the extrachromosomal palindrome ribosomal RNA genes of Dictyostelium. Confirming our earlier results with micrococcal nuclease (1,2), MPE.Fe(II) digested the coding region of rapidly transcribing rRNA genes as a smear, indicating the absence or severe disruption of nucleosomes, whereas in slowly transcribing rRNA genes, a nucleosomal ladder was produced. In the central non-transcribed spacer region of the palindrome, MPE.Fe(II) digestion resulted in a normal nucleosomal repeat, whereas micrococcal nuclease gave a complex banding pattern. The difference is attributed to the lower sequence specificity of MPE.Fe(II) compared to micrococcal nuclease. In the terminal region of the palindrome, however, both substances gave a complex chromatin digestion pattern. In this region the DNA appears to be packaged in structures strongly positioned with respect to the underlying DNA sequence.     

Fractionation of detergent lysates of cells by ammonium sulphate-induced phase separation

A procedure is described for fractionating detergent lysates of cells based on the ability of (NH4)2SO4 to induce phase separation of detergents such as Triton X-100, sodium deoxycholate, and sodium cholate, into detergent-rich and detergent-depleted phases. An analysis of six murine lymphocyte cell surface molecules revealed that the partitioning in Triton X-100 of each molecule was highly dependent upon the (NH4)2SO4 concentration, each antigen partitioning into the detergent-rich phase at a defined salt concentration. In contrast, none of the six molecules appeared in the detergent-rich phase of a Triton X-114 phase separation, even though two of the molecules, namely Ly-2/3 and L3T4, are well-characterized integral membrane proteins. It was also observed that (NH4)2SO4 resulted in the partitioning of many nonmembrane proteins into the detergent-rich phase, indicating that the procedure can be used to fractionate all cellular proteins. By judicious choice of (NH4)2SO4 concentrations, precipitation of cellular proteins at two different (NH4)2SO4 concentrations, and combining the method with subcellular fractionation prior to detergent solubilization, substantial enrichment and concentration of particular cellular proteins could be achieved.     

The influence of side chain modifications of the heme moiety on prosthetic acceptance and function of rat hepatic cytochrome P-450 and tryptophan pyrrolase

The relative potential of various structural isomers (III, XIII) and various 2,4-side chain modified analogs of heme (iron-protoporphyrin IX) to incorporate into rat liver hemoproteins, cytochrome P-450(s), and tryptophan pyrrolase was examined. Such assessments for hepatic cytochrome P-450 relied on generation of reconstitutible apocytochrome(s) P-450 by suicidal alkylation of the existing prosthetic heme moiety by allylisopropylacetamide (AIA) in vivo. Subsequent replacement of the prosthetic heme was brought about by incubating the apocytochrome(s) P-450-enriched preparations with a particular heme isomer or analog. Structure-function relationships of the reconstituted isozymes were assessed in microsomal preparations by monitoring cytochrome P-450 content (structure) and its mixed function oxidase activity (function). In parallel, the relative ability of these heme isomers and analogs to functionally constitute hepatic tryptophan pyrrolase was also assessed by monitoring the relative increase in holoenzyme activity when preparations deliberately enriched in constitutible apoenzyme were incubated with each of these compounds. The findings reveal that 2,4-side chain modifications on the heme IX skeleton markedly influence the function of the constituted hemoproteins possibly by affecting their structural assembly through steric, electronic, and/or hydrophobic interactions with the corresponding apoproteins. Furthermore, these studies not only reveal that the structural specifications of the active prosthetic site of rat liver cytochrome P-450(s) differ from those of tryptophan pyrrolase, but also that the structural specifications of these mammalian hemoproteins for their prosthetic heme differ considerably from those reported for their bacterial counterparts.     

Studies on senescing tobacco leaf disks with special reference to peroxidase

Summary Senescing tobacco leaf disks were treated with hydroxyproline (hypro). Chlorophyll breakdown and -amino-nitrogen increase were partially inhibited, but the decrease in soluble protein was stimulated. The normal increase in absolute acid-phosphatase activity was reversed, but the increase in peroxidase activity occurred although after a lag phase. Kinetin inhibition of senescence was reversed by hypro and kinetin actually reinforced the effects of hypro. Proline only partially overcame the effects of hypro plus kinetin. In the presence of kinetin, proline considerably stimulated the increase in peroxidase activity.Discussed are the way in which hypro may be acting, and the possibility of peroxidase being involved in proline hydroxylation.     

Ability of different chemically modified heparins to potentiate the biological activity of heparin-binding growth factor 1: lack of correlation with growth factor binding.

A range of chemically modified heparins was examined for their ability to bind heparin-binding growth factor 1 (HBGF-1; acidic fibroblast growth factor) and potentiate the in vitro mitogenic and neurotrophic activity of HBGF-1. It was found that carboxyl-reduced heparin bound HBGF-1 as effectively as the native heparin molecule. Totally desulfated heparin and N-desulfated heparin lack HBGF-1-binding capacity, and substitution of the exposed amino group with acetyl or acetoacetyl groups only partially restored binding capacity, indicating that N-sulfates only play a limited role in growth factor binding. However, the failure of totally desulfated, N-resulfated heparin to interact with HBGF-1 demonstrated that N-sulfates alone are insufficient and ester sulfates are absolutely essential for HBGF-1 binding. In contrast, the ability of the modified heparins to potentiate the mitogenic activity of HBGF-1 correlated only to a limited extent with their affinity for HBGF-1. Thus, the carboxyl-reduced molecule which displayed similar affinity for HBGF-1 as native heparin was consistently less potent in augmenting mitogenesis. Similarly, the N-acetylated and the N-acetoacetylated species, which had much lower affinity for HBGF-1 than the carboxyl-reduced molecule, conferred similar biological activity to HBGF-1 whereas N-desulfated heparin, which was unable to bind growth factor, potentiated the mitogenic activity of HBGF-1 for both 3T3 and HUVE cells. In contrast, the neurotrophic activity of HBGF-1 was potentiated by modified heparin species which failed to bind HBGF-1 and were without activity in the mitogenic assays.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitors of sterol synthesis. Chemical syntheses, properties and effects of 4,4-dimethyl-15-oxygenated sterols on sterol synthesis and on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in cultured mammalian cells

The chemical syntheses of a number of 4,4-dimethyl substituted 15-oxygenated sterols have been pursued to permit evaluation of their activity in the inhibition of the biosynthesis of cholesterol and other biological effects. Described herein are the first chemical syntheses of 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-3 beta-ol-15-one, 3 beta,15 alpha-diacetoxy-4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene, 3 beta-acetoxy-4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-15 beta-ol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 beta-diol, 4,4-dimethyl-14 alpha-ethyl-5 alpha-cholest-7-en-15 alpha-ol-3-one, 3 beta-benzoyloxy-4,4-dimethyl-5 alpha-cholest-8(14)-ene-7 alpha,15 alpha-diol, 7 alpha,15 alpha-diacetoxy-3 beta-benzoyloxy-4,4-dimethyl-5 alpha-cholest-8(14)-ene, 4,4-dimethyl-5 alpha-cholest-8(14)-en-3 beta-ol-15-one and 3 beta,7 alpha,15 alpha-tri-o-bromobenzoyloxy-5 alpha-cholest-8(14)-ene. Also prepared for use in the biological experiments were 4,4-dimethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol, 4,4-dimethyl-5 alpha-cholest-8-ene-3 beta,15 alpha-diol and 4,4-dimethyl-5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol. The effects of twelve 4,4-dimethyl substituted 15-oxygenated sterols and of four 4,4-dimethyl substituted 32-oxygenated sterols on sterol synthesis and on the level of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity were evaluated in mouse L cells. With the exception of 4,4-dimethyl-5 alpha-cholest-8(14)-ene-3 beta,7 alpha,15 alpha-triol, all of the 4,4-dimethyl substituted 15-oxygenated sterols caused a 50% inhibition of sterol synthesis at less than 10(-6) M and six of the 4,4-dimethyl substituted 15-oxygenated sterols caused a 50% inhibition of sterol synthesis at less than 10(-7) M. 4,4-Dimethyl-14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta,15 alpha-diol caused a 50% decrease in sterol synthesis at 10(-8) M. The potencies of the 4,4-dimethyl substituted 15-oxygenated and C-32-oxygenated sterols with respect to inhibition of sterol synthesis and suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity have been compared with those of the corresponding sterols lacking the 4,4-dimethyl substitution.     

The roles of cell-wall acidification and proton-pump stimulation in auxin-induced growth: studies using monensin

The carboxylic ionophore, monensin, rapidly induced cell-wall acidification and a decrease in cytosolic pH when added to maize coleoptiles at low external pH and Na⁺ concentration. Elongation growth at rates equivalent to those obtained with indole-3-acetic acid was induced for about 1 h. Stimulation of the outwardly directed proton pump apparently occurred, since under the same conditions monensin induced membrane hyperpolarization of maize root rhizodermis cells. When the external pH was high (>8) and Na⁺ present, monensin treatment caused only minimal changes in membrane potential and cytosolic pH. Although the ionophore transported protons out of the cell, resulting in cell-wall acidification, no elongation growth occurred. However, under identical conditions, indole-3-acetic acid dit induce growth. The data indicates that stimulation of the outwardly directed electrogenic proton pump rather than the subsequent acidification of the cell wall is vital for the induction of elongation growth.Abbreviations CFA₂ 6-carboxyfluorescein diacetate - FA₂ fluorescein diacetate - Hepes 4-(2-hydroxyethyl-1-piperazinepropanesulfonic acid - IAA indole-3-acetic acid - Mes 2-(N-morpholino) ethanesulfonic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Termination of reproduction in nonhuman and human female primates

We examined demographic records from 13 captive primate species and a human population to determine age-related changes in female reproduction. In most species age-specific fertility declined and interbirth intervals increased with age. Using an operational definition of termination of reproduction based on individual variance in interbirth intervals, a proportion of females in most nonhuman species had terminated reproduction before death. Compared to other primates, a greater proportion of chimpanzees and human females ceased reproduction, and humans, in particular, were reproductively inactive for relatively longer than would be expected from their body weight. These empirical data quantify the extent of reproductive termination and thereby extend hitherto anecdotal accounts of this phenomenon in primates.