Modifications to histones immediately after synthesis

We have studied modifications occurring on histones immediately after synthesis. The labeling period was sufficiently brief such that the time interval between synthesis in the cytoplasm and deposition onto DNA can be easily detected. In this way it is possible to observe modifications occurring within seconds after arrival of the newly synthesized histone molecule in the nucleus. Histone F_2a1 is rapidly and extensively modified (in hepatoma tissue culture cells this is primarily by acetylation) so that the newly synthesized material appears primarily in the diacetyl modified form. The histone returns to a steady-state level of acetylation during the ensuing 30 minutes and this level is maintained throughout the lifetime of the cell. F₃ shows a somewhat similar pattern, except that it initially is less modified before it shifts to a steady-state level of modification. The lysinerich histone, F₁ is admitted to the nucleus with a modest level of phosphorylation. During the subsequent 60-minutes phosphorylation proceeds rapidly until the extent of phosphorylation typical of rapidly dividing cells is reached. This level of phosphorylation is then maintained in a steady-state against the action of a phosphatase. The rate of phosphorylation during the initial 60 minutes exceeds that at a later time by a factor of ~ ten. We argue that this observation accounts for previous observations of ³²P incorporation during G₁ and S phase and in the presence of inhibitors of DNA and histone synthesis.     

Initial steps in the large-scale purification of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid polymerase

Histones from exponential and stationary-phase mouse L-cells were quantitated after acrylamide gel electrophoresis in order to investigate cell cycle-dependent changes in the mode of binding of the various fractions in chromatin. By introducing various concentrations of citrate and divalent cations in the medium used for cell lysis and isolation of nuclei prior to histone extraction it was possible to demonstrate that certain histone fractions are preferentially retained in either exponential or stationary-phase nuclei. Differential retention of lysine-rich F₁ was most evident when the lysing medium contained 1 mm Mg²⁺ and Ca²⁺ and 5 mm citrate (pH 2.75). In these conditions twice as much F₁ is retained in stationary as in exponential nuclei. Differential retention of arginine-rich histones was most evident when the lysing medium contained 10 mm Mg²⁺ and Ca²⁺ and no citrate. In these conditions more F_{2a 1} is retained in exponential than in stationary nuclei while the opposite is true for F₃. However, the total amount of arginine-rich fractions (F_{2a 1} + F₃) retained was found to be the same in both cell phases. The results are discussed in relation to known structural features of the histones.     

A novel fluorescence chamber for the determination of volume changes in human CaSki cell cultures attached on filters

The objective of the study was to test the hypothesis that, in the cultured human cervical epithelium, CaSki, the effect of calcium mobilizing agents on transepithelial electrical conductance (G_TE), is the result of cell volume decrease. CaSki cells attached on filters were loaded with fura-2, and measurements of fluorescence at the isosbestic wavelength 360 nm (excitation/emission [F_360/510]) were made in a newly designed fluorescence chamber; this design allowed us also to determine changes in cytosolic calcium ([Ca²⁺]_i). The experimental conditions were similar to those used to measure changes in paracellular permeability in the Ussing chamber, and they enabled us to compare the time-course of changes in [Ca²⁺]_i, in F_360/510, and in G_TE. Hypertonicity increased, and hypotonicity decreased F_360/510 and G_TE, without having an effect on [Ca²⁺]_i, and the changes in F_360/510 and in G_TE correlated linearly. Metabolism, bleaching, and extrusion of intracellular fura-2 were minimal, indicating that the changes in F_360/510 reflect changes in dye concentration. Hypertonicity decreased, and hypotonicity increased the size of dispersed CaSki cells, suggesting that osmolarity-induced changes in F_360/510 reflect changes in size of the attached cells. Ionomycin increased [Ca²⁺]_i, F_360/510, and G_TE, but the increases in [Ca²⁺]_i preceded those in F_360/510 and G_TE. The calcium chelator BAPTA blocked the ionomycin-induced increase in [Ca²⁺]_i, F_360/510, and in G_TE. Preincubation with 4-acetamido-4′isothiocyanatostilbene-2,2′disulfonic acid (SITS) augmented the ionomycin-induced increase in [Ca²⁺]_i, but blocked the increases in F_360/510 and in G_TE. Pretreatment of cells with hypertonic solution abrogated the increases in F_360/510 and in G_TE in response to ionomycin, but had little effect on the ionomycin-induced increase in [Ca²⁺]_i. On the basis of these results we suggest that the ionomycin-induced increase in G_TE is mediated by [Ca²⁺]_i-dependent chloride secretion and osmotic water loss.     

Retinoic acid-promoted expansion of total cellular ATP pools in 3T3 cells can mediate its stimulatory and growth inhibitory effects

Exposure of Swiss 3T3 cells to micromolar quantities of β-all-trans-retinoic acid (RA) results in either inhibition of growth or stimulation of cellular responsiveness to mitogens, depending on the length of treatment. Inhibition of growth is produced by treatment of the cells with RA for at least 48 hours. The total cellular pools of adenosine 5′-triphosphate (ATP) are markedly increased after 48-hour RA treatment and dose dependence studies show a correlation between the expanded ATP pools and the inhibitory effects. The expansion of total cellular ATP pools by retinoic acid occurs throughout the cell cycle and parallels the cell cycle-dependent fluctuations in total cellular ATP pools of untreated cells. Studies of [³H]thymidine incorporation and labeling indices in intact cells and [³H]dTTP incorporation and labeling indices in isolated nuclei of RA-treated and control cultures suggest that cellular acid-soluble nucleotide pools mediate the inhibition of DNA replication in the 48-hour-RA-treated cells. The stimulatory activity of RA for mitogenic responsiveness is demonstrated by treatment of G₀/G₁-arrested 3T3 cells with micromolar levels of RA for a maximum of 18 hours resulting in the potentiation of phorbol myristate acetate (PMA)-stimulated transition into S phase of the cell cycle. Marked increases in total cellular ATP and UTP pools are produced by 18-hour treatment of G₀/G₁-arrested cells with RA, before their exposure to PMA.     

Effect of the natural ATPase inhibitor on the binding of adenine nucleotides and inorganic phosphate to mitochondrial F1-ATPase

(1) Incubation of the beef heart mitochondrial ATPase, F₁ with Mg-ATP was required for the binding of the natural inhibitor, IF₁, to F₁ to form the inactive F₁-IF₁ complex. When F₁ was incubated in the presence of [¹⁴C]ATP and MgCl₂, about 2 mol ¹⁴C-labeled adenine nucleotides were found to bind per mol of F₁; the bound ¹⁴C-labeled nucleotides consisted of [¹⁴C]ADP arising from [¹⁴C]ATP hydrolysis and [¹⁴C]ATP. The ¹⁴C-labeled nucleotide binding was not prevented by IF₁. These data are in agreement with the idea that the formation of the F₁-IF₁ complex requires an appropriate conformation of F₁. (2) The ¹⁴C-labeled adenine nucleotides bound to F₁ following preincubation of F₁ with Mg-[¹⁴C]ATP could be exchanged with added [³H]ADP or [³H]ATP. No exchange occurred between added [³H]ADP or [³H]ATP and the ¹⁴C-labeled adenine nucleotides bound to the F₁-IF₁ complex. These data suggest that the conformation of F₁ in the isolated F₁-IF₁ complex is further modified in such a way that the bound ¹⁴C-labeled nucleotides are no longer available for exchange. (3) ³²P_i was able to bind to isolated F₁ with a stoichiometry of about 1 mol of P_i per mol of F₁ (Penefsky, H.S. (1977) J. Biol. Chem. 252, 2891–2899). There was no binding of ³²P_i to the F₁-IF₁ complex. Thus, not only the nucleotides sites, but also the P_i site, are masked from interaction with external ligands in the isolated F₁-IF₁ complex.     

Expression of the μ-Opioid Receptor in CHO Cells: Ability of μ-Opioid Ligands to Promote α-Azidoanilido[32P]GTP Labeling of Multiple G Protein α Subunits

Abstract: The identities of heterotrimeric G proteins that can interact with the μ-opioid receptor were investigated by α-azidoanilido[³²P]GTP labeling of α subunits in the presence of opioid agonists in Chinese hamster ovary (CHO)-MORIVA3 cells, a CHO clone that stably expressed μ-opioid receptor cDNA (MOR-1). This clone expressed 1.01 × 10⁶μ-opioid receptors per cell and had higher binding affinity and potency to inhibit adenylyl cyclase for the μ-opioid-selective ligands [d -Ala²,N-MePhe⁴,Gly-ol]-enkephalin and [N-MePhe³,d -Pro⁴]-morphiceptin, relative to the δ-selective opioid agonist [d -Pen²,d -Pen⁵]-enkephalin or the κ-selective opioid agonist U-50,488H. μ-Opioid ligands induced an increase in α-azidoanilido[³²P]GTP photoaffinity labeling of four Gα subunits in this clone, three of which were identified as G_i3α, G_i2α, and G_o2α. The same pattern of simultaneous interaction of the μ-opioid receptor with multiple Gα subunits was also observed in two other clones, one expressing about three times more and the other 10-fold fewer receptors as those expressed in CHO-MORIVA3 cells. The opioid-induced increase of labeling of these G proteins was agonist specific, concentration dependent, and blocked by naloxone and by pretreatment of these cells with pertussis toxin. A greater agonist-induced increase of α-azidoanilido[³²P]GTP incorporation into G_i2α (160–280%) and G_o2α (110–220%) than for an unknown Gα (G_?α) (60%) or G_i3α (40%) was produced by three different μ-opioid ligands tested. In addition, slight differences were also found between the ability of various μ-opioid agonists to produce half-maximal labeling (ED₅₀) of any given Gα subunit, with a rank order of G_i3α > G_o2α > G_i2α = G_?α. In any case, these results suggest that the activated μ-opioid receptor couples to four distinct G protein α subunits simultaneously.     

Replication-independent nucleosome exchange is enhanced by local and specific acetylation of histone H4

We used a novel single-cell strategy to examine the fate of histones during G₂-phase. Consistent with previous results, we find that in G₂-phase, the majority of nuclear histones are assembled into chromatin, whereas a small fraction comprises an unassembled pool. Small increases in the amount of histones within the free pool affect the extent of exchange, suggesting that the free pool is in dynamic equilibrium with chromatin proteins. Unexpectedly, acetylated H4 is preferentially partitioned to the unassembled pool. Although an increase in global histone acetylation did not affect overall nucleosome dynamics, an H4 containing lysine to glutamine substitutions as mimics of acetylation significantly increased the rate of exchange, but did not affect the acetylation state of neighbouring nucleosomes. Interestingly, transcribed regions are particularly predisposed to exchange on incorporation of H4 acetylation mimics compared with surrounding regions. Our results support a model whereby histone acetylation on K8 and K16 specifically marks nucleosomes for eviction, with histones being rapidly deacetylated on reassembly.     

Purification and characterisation of 1F-fructosyltransferase from the roots of asparagus (asparagus officinalis L.)

A fructosyltransferase that transfers a terminal d-fructosyl group from a (2→1)-β-linked fructosaccharide to HO-1 of another d-fructosyl group has been purified from an extract of asparagus roots by successive chromatography with DEAE-cellulose, octyl-Sepharose, Sephadex G-200, and raffinose-coupled Sepharose 6B. The disc-electrophoretically homogeneous enzyme was free from β-d-fructofuranosidase, sucrose:sucrose 1-fructosyltransferase, and 6^G-frutosyltransferase activity, and catalysed the d-fructosyl transfer from 1-kestose more rapidly to saccharides of the neokestose series [1^F(1-β-d-fructofuranosyl)_m-6^G(1-β-d-fructofuranosyl)_nsucrose] than to those of the 1-kestose series [1^F(1-β-d-fructofuranosyl)_nsucrose]. The enzyme was tentatively termed 1^F-fructosyltransferase. The general properties of the enzyme were as follows: mol. wt., ~64,000; optimum pH, ~5.0; stable at pH 5.0–5.5 at 45° for 20 min; stable at 30–45° for 10 min; inhibited by Hg²⁺, p-chloromercuribenzoate, and Ag⁺.     

Acetylation of histones of rat testis.

When [1-¹⁴C]acetate was injected into rats intratesticularly in the presence of cycloheximide to inhibit protein synthesis, the label was incorporated into histone fractions F2a1 and F3 and into non-histone chromosomal proteins of each of the following stages of spermatogenesis: spermatogonia-preleptotene spermatocytes, leptotene-zygotene-pachytene-diplotene primary spermatocytes, and spermatids. Acetylation of histones was particularly active in the spermatid stages. There was no significant incorporation of acetate into the lysine-rich histone fractions F1 and X₁.In early periods of in vivo incorporation of [³H]amino acids into histones the acetylated histone F2a1 fractions had higher specific activities than the main band of F2a1, but with the passage of time the label moved into the principal band to the extent that specific activities in the acetylated and principal bands were approximately equal at 6 days. However, at 24–36 days the specific activities were again higher in the acetylated bands than in the principal band of F2a1. These data support the conclusions of Candido, Louie, and Dixon, from experiments with trout testis, that acetylation of histone F2a1 may be important in the process of combination of this protein with DNA in chromatin at the spermatogonia-primary spermatocyte stage and also in the subsequent removal of this histone for replacement by protamines at the spermatid stage.[³H]Amino acids were incorporated into histone fractions X₁ and F1 at approximately equal rates, and there was no evidence that one of these fractions was a precursor of the other.Chromatin of the seminiferous epithelial cells of rat testis has a firmly bound acetylase which catalyzes the in vitro acetylation of histones F3 and F2a1 by acetyl CoA.     

Measurement of Cerebral Glucose Utilization Using Washout After Carotid Injection in the Rat

Abstract: The carotid injection technique, used previously to quantitate the kinetics of blood-brain barrier transport of metabolic substrates, may be modified to analyze the rate of cerebral glucose utilization. A 0.2-ml solution of [¹⁴C]glucose (G_F) and [³H]methylglucose (M), an internal reference, is rapidly injected into the carotid artery, followed by microwave fixation of brain at various times up to 4 min after injection. The brain radioactivity is separated into a fraction containing neutral hexoses (G_F and M) and a fraction containing metabolites of glucose. The G_F/M ratio is related to the rate constant (k₃) of brain glucose utilization by the simple, linear equation: In(G_F/M) = In(G_F°/M°) –k₃t, where G_F°/M°= the brain uptake index of glucose, relative to methylglucose, at 5-15 s after injection, and t= the time after carotid injection, e.g., 1–4 min. It is assumed that (a) the rate of influx due to recirculation of label is minimal during the 4-min circulation period; and (b) the rate constants of glucose efflux (k₂) and methylglucose efflux (k₂*) are identical. Independent estimates of k₂ and k₂* showed these parameters to be identical: k₂= 0.14 + 0.08 min-I; k₂*= 0.14 ± 0.02 min-I. A logarithmic plot of G_F/M ratios versus time was linear (r = 0.99), and was described by the slope k₂= 0.21 ± 0.02 min^?1. Assuming glucose is uniformly distributed in brain, then the glycolytic rate = k₃× brain glucose = (0.21 min^?1) (2.6 μmol g^?1) = 0.55 μmol min^?1 g^?1 for the cortex of the barbiturate-anesthetized rat. These studies provide the basis for a simple method of measurement of regional brain glycolysis that does not require either the use of correction factors, e.g., the lumped constant, or the use of differentially labeled glucose.     

High-affinity binding sites for prostaglandin E on human lymphocytes.

Binding sites on human lymphocytes for prostaglandins were examined by incubating cells with [³H]prostaglandin (PG) A₁, E₁, E₂, F_1α, and F_2α. Specific reversible binding for [³H]PGE₁ and E₂ was found with a K_d of ~2 × 10^?9M and a B max of ~200 binding sites per cell, assuming uniform distribution. We detected no specific binding of [³H]PGA₁, F_1α, or F_2α to lymphocytes. Also, the addition of 10- to 1000-fold greater amounts of unlabeled PGA, F_1α, or F_2α did not inhibit the binding of [³H]PGE. The time course of [³H]PGE binding appeared to be bimodal with one component complete within 5 min at 37 °C and another component of binding increasing over a 40-min incubation. We feel that the rapid component of binding may represent cell surface receptors for PGE while the slower component may represent a specific uptake mechanism for PGE into the cell. Glass adherent cells had fewer binding sites than nonadherent cells. Preincubation of the cells overnight resulted in a loss of binding sites.     

Specific changes in the biosynthesis and acetylation of nucleosomal histones in the early stages of embryogenesis of sea urchin.

Histones of the sea urchin Strongylocentrotus droebachiensis were labelled in vivo with [¹⁴C]protein hydrolysate and [¹⁴C]sodium acetate at the developmental stages of blastula, gastrula and pluteus. They were further resolved by electrophoresis in 15% polyacrylamide gels in the presence of 0.9 M acetic acid, 8 M urea as well as in 20% polyacrylamide gels containing the same concentrations of acetic acid and urea, but in addition, 4.3 mM Triton X-100. On comparison of electrophoretic patterns with autoradiograms it was shown that during the early stages of differentiation the synthesis of several new subfractions of some of the histones takes place, namely of two of H1, of two of H2b and of nine of H2a. At the same time the electrophoretic pattern of the arginine-rich histones remained constant, H3 consisting of three and H4 of four subfractions. The basic reason for this heterogeneity seems to be the specific post-synthetic acetylation of the histones. At the blastula stage all the newly synthesized H4 molecules are equally acetylated, while the histone H3 molecules are acetylated to varying extents. After gastrulation both H3 and H4 are also subject to a different level of acetylation.     

Effects of p21Cip1/Waf1 at Both the G1/S and the G2/M Cell Cycle Transitions: pRb Is a Critical Determinant in Blocking DNA Replication and in Preventing Endoreduplication

It has been proposed that the functions of the cyclin-dependent kinase inhibitors p21^Cip1/Waf1 and p27^Kip1 are limited to cell cycle control at the G₁/S-phase transition and in the maintenance of cellular quiescence. To test the validity of this hypothesis, p21 was expressed in a diverse panel of cell lines, thus isolating the effects of p21 activity from the pleiotropic effects of upstream signaling pathways that normally induce p21 expression. The data show that at physiological levels of accumulation, p21, in addition to its role in negatively regulating the G₁/S transition, contributes to regulation of the G₂/M transition. Both G₁- and G₂-arrested cells were observed in all cell types, with different preponderances. Preponderant G₁ arrest in response to p21 expression correlated with the presence of functional pRb. G₂ arrest was more prominent in pRb-negative cells. The arrest distribution did not correlate with the p53 status, and proliferating-cell nuclear antigen (PCNA) binding activity of p21 did not appear to be involved, since p27, which lacks a PCNA binding domain, produced similar arrest Bs. In addition, DNA endoreduplication occurred in pRb-negative but not in pRb-positive cells, suggesting that functional pRb is necessary to prevent DNA replication in p21 G₂-arrested cells. These results suggest that the primary target of the Cip/Kip family of inhibitors leading to efficient G₁ arrest as well as to blockade of DNA replication from either G₁ or G₂ phase is the pRb regulatory system. Finally, the tendency of Rb-negative cells to undergo endoreduplication cycles when p21 is expressed may have negative implications in the therapy of Rb-negative cancers with genotoxic agents that activate the p53/p21 pathway.     

The effect of isoproterenol and hydroxyurea on the presence of ubiquitin and protein A24 in the rat salivary gland

The in vivo administration of hydroxyurea for 12 h counteracts DNA synthesis and cell cycling stimulated by 72 h of isoproterenol treatment in rat salivary gland, as determined by fluorescence-activated flow cytometry. Hydroxyurea has little effect on [³H]leucine incorporation (protein synthesis) of the nuclear proteins soluble in 0.35 M NaCl, when examined by polyacrylamide gel chromatography and autoradiography from electro-statically sorted nuclei of (G₀+G₁) and (G₂+M) phases of the in vivo cell cycle. Differential incorporation of [³H]leucine into nuclear proteins was observed during various phases of the cell cycle. Proteins ‘X’ and ‘Z’, observed in stained gel chromatographs of the 0.35 M NaCl-soluble nuclear proteins, were identified by biochemical analyses as ubiquitin and protein A₂₄, respectively. Ubiquitin appeared transiently while A₂₄ increased in gel chromatograms concomitant with progressive quiescence of the salivary gland induced by hydroxyurea.     

Purification and characterisation of 6G-fructosyltransferase from the roots of asparagus (Asparagus officinalis L.)

A fructosyltransferase that catalyses the transfer of the terminal (2 → 1)-β-linked d-fructosyl group of fructo-oligosaccharides [1^F(1-β-d-fructofuranosyl)_msucrose, m > 0] to HO-6 of the glucosyl group of similar saccharides [1^F(1-β-d-fructofuranosyl)_nsucrose, n > 0] has been purified (760-fold) from an extract of the roots of asparagus (Asparagus officinalis L.) by successive fractionation with ammonium sulfate, treatment with calcium phosphate gel, and then chromatography on octyl-Sepharose, DEAE-cellulose, Sephadex G-200, and raffinose-coupled Sepharose 6B. The enzyme, tentatively termed 6^G-fructosyltransferase, was homogeneous in disc electrophoresis, had a mol. wt. of ~69,000 and an optimum pH of ~5.5, was stable at pH 5.0–6.0 on heating for 20 mins at 45° and for 10 min at 20–37°, and was inhibited by Hg²⁺, p-chloromercuribenzoate, and Ag⁺.     

cAMP and PMA enhance the effects of IGF-I in the proliferation of endometrial adenocarcinoma cell line HEC-1-A by acting at the G1 phase of the cell cycle

The present study was undertaken to determine whether endometrial cancer cell line HEC-1-A differ from nontransformed cells, in that the cAMP and protein kinase C pathways may enhance IGF-I effects in mitogenesis by acting at the G₁ phase of the cell cycle instead of G₀. Immunofluorescence staining of HEC-1-A cells using the proliferating cell nuclear antigen (PCNA) monoclonal antibody and flow cytometric analysis determined that HEC-1-A cells do not enter the G₀ phase of the cell cycle when incubated in a serum-free medium. Approximately 51% of the cells were in G₁, 12% were in S and 37% in G₂ phase of the cell cycle prior to treatment. Forskolin and phorbol-12-myristate 13-acetate (PMA) were used to stimulate cAMP production and protein kinase C activity, respectively. IGF-I, forskolin and PMA each increased (P <0.01) [³H]-thymidine incorporation in a dose and time dependent manner. The interaction of forskolin and PMA with IGF-I was then determined. Cells preincubated with forskolin or PMA followed by incubation with IFG-I incorporated significantly more (P <0.01) [³H]-thymidine into DNA than controls or any treatment alone. It is concluded that forskolin and, to a lesser extent, PMA exert their effect at the G₁ phase of the cycle to enhance IGF-I effects in cell proliferation.     

Expression of the mitochondrial genome in HeLa cells. XXI. Mitochondrial protein synthesis during the cell cycle

Chloramphenicol sensitive [³H]leucine incorporation into protein (due to mitochondrial protein synthesis) in synchronized HeLa cells has been found to continue throughout interphase, its rate per cell approximately doubling from the G₁ to the G₂ phase. This increase in the rate of [³H]leucine incorporation during the cycle does not seem to parallel closely the increase in cell mass. In fact, the observations made on cultures incubated at 34.5 °C, where the G₁ and S phases are better resolved than at 37 °C, indicate that the rate remains constant during the G₁ phase, and starts to accelerate with the onset of nuclear DNA synthesis. Correspondingly, on a per unit mass basis, there appears to be a slight decline in the rate of [³H]leucine incorporation into protein during the G₁ phase, which is compensated by an increase in the early S phase. No significant variations were observed in the mitochondrial leucine pool labeling during the cell cycle; therefore, the observed pattern of [³H]leucine incorporation into protein should reflect fairly accurately the behavior of mitochondrial protein synthesis. Evidence has been obtained indicating a depression in the rate of incorporation of [³H]leucine into protein in mitochondria of mitotic cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the products of mitochondrial protein synthesis has not revealed any differences in the size distribution of the proteins synthesized in the various portions of the cell cycle.     

THE SYNTHESIS OF ACIDIC CHROMOSOMAL PROTEINS DURING THE CELL CYCLE OF HELA S-3 CELLS : I. The Accelerated Accumulation of Acidic Residual Nuclear Protein before the Initiation of DNA Replication

The synthesis and accumulation of acidic proteins in the tightly bound residual nuclear fraction goes on throughout the cell cycle of continuously dividing populations of HeLa S-3 cells; however, during late G₁ there is an increased rate of synthesis and accumulation of these proteins which precedes the onset of DNA synthesis. Unlike that of the histones, whose synthesis is tightly coupled to DNA replication, the synthesis of acidic residual nuclear proteins is insensitive to inhibitors of DNA synthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of acidic residual nuclear proteins shows different profiles during the G₁, S, and G₂ phases of the cell cycle. These results suggest that, in contrast to histones whose synthesis appears to be highly regulated, the acidic residual proteins may have a regulatory function in the control of cell proliferation in continuously dividing mammalian cells.     

Isolation and Identification of Fructo-oligosaccharides in Roots of Asparagus (Asparagus officinalis L.)

Eight fructo-oligosaccharides were isolated from purified oligosaccharide fractions of the roots of Asparagus officinalis L. (Liliaceae). By examination of constituent sugars, gas-liquid-chromatographic analysis of methyl derivatives, and investigation of partial acid hydrolyzates and products of β-fructofuranosidase action, they were confirmed to be 1^F(1-β -fructofuranosyl)_n sucrose [n = 1 (1-kestose), 2 (nystose), and 3], 6^G (1-β-fructofuranosyl)_n sucrose [n = 1 (neokestose), 2, and 3], 1^F,6^G-di-β-fructofuranosyl sucrose, and a new pentasaccharide 1^F (1-β-fructofuranosyl)₂-6^G-β-fructofuranosyl sucrose.