Isolation of a rat ventral prostate chromatin fraction exhibiting a singular melting profile

Rat ventral prostate chromatin, prepared by a gentle procedure in which marked shearing forces were avoided, was separated by sucrose gradient centrifugation into a quantitatively minor less dense (L) fraction, and 2 heavier components, (H₁) and (H₂). The (L) fraction, comprising 5–15 percent of the totäl chromatin DNA, had a unique melting profile, with a Tm_app. of 59°, compared to 82.5°, 85° and 80.5° for total, (H₁) and (H₂) chromatin. (L) chromatin has a number of other properties expected of “euchromatin”. This approach to the preparation of chromatin has been used successfully with other tissues such as liver.     

Solubilization and characterization of RNA polymerase from a higher plant

RNA polymerase has been solubilized from sugar beet chromatin. With calf thmus or sugar beet DNA as template enzyme activity was linear with respect to protein concentration and required the presence of all four nucleoside triphospahates, added DNA and divalent metal ions. The enzyme exhibited a sharp Mn²⁺ optimum of 1·25 mM and a Mg²⁺ optimum at 10mM. The Mn²⁺/Mg²⁺ activity ratio (activity at optimum concentrations) was 2·0 with an optimum salt concentration of 50 mM. Based on data including inhibition with α-amanitin (0·025 μg/ml), the majority of the total activity appeared to be RNA polymerase I. Subsequent fractionation by DEAE-Sephadex column chromatography resulted in one peak of activity eluted with 0·18 M (NH₄)₂SO₄.     

Studies on the mode of action of calciferol: Subcellular localization of 1,25-dihydroxyvitamin D3 in chicken parathyroid glands

As a further means of evaluating 1,25-dihydroxyvitamin D₃-parathyroid gland interaction and its relation to calcium homeostasis, a comparative study of the subcellular localization of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]in the parathyroid glands, intestinal mucosa, kidney, and liver of rachitic chickens has been carried out. Only in the chromatin fraction from parathyroids and intestinal mucosa could there be demonstrated selective and specific localization of the 1,25(OH)₂D₃. The chromatin-bound picomoles of 1,25(OH)₂D₃ (per gram of tissue) was in the ratio (mucosa:parathyroids:kidney:liver) of 1.0:0.23:0.11:0.17 2 h after an intracardial injection of 290 pmol of [³H]1,25(OH)₂D₃. This same ratio after a 30-min (23 °C) homogenate incubation with 1 × 10^?8m [³H]1,25(OH)₂D₃ was 1.0:1.0:0.10:0.03. Analogous results were obtained when reconstituted chromatin and cytosol fractions from the different tissues were compared for chromatin localization efficiency. This chromatin localization of 1,25(OH)₂D₃ in the parathyroid glands was temperature dependent. In addition, parathyroid glands were found to contain 3.0–3.5 S cytoplasmic and KCl-extractable chromatin receptors specific for 1,25(OH)₂D₃.     

Presence of prostaglandins E2 and A2 in canine gastric secretions

The presence of prostaglandins (PGs) was determined in gastric juice obtained from 3 conscious dogs, provided with a chronic gastric fistula. Outputs of acid (mequiv min^?1) and PGs (pg min^?1) were measured in gastric secretions stimulated by pentagastrin (100 or 200 ng kg^?1min^?1). Prostaglandin activity was estimated, after extraction and thin layer chromatography, by radioimmuno-assay of the PGB formed by treatment with alkali. Tritiated PGs were added to gastric juice for the purpose of correcting for PGs recovery. Using this method, the minimum mass of PGB which could be satisfactorily distinguished from zero was 25 pg. Prostaglandins A₂ and E₂ were present in pentagastrin-activated gastric secretions and averaged (mean ± SE, n = 8) 200.7 ± 18.1 and 260.1 ± 18.0 pg min^?1 respectively. The identity of PGA₂ and PGE₂ was confirmed by gas liquid chromatography combined with mass spectrometry. The amount of PGE₂ converted to PGA₂ during extraction, separation and conversion procedures was estimated from the amount of [³H] PGA₂ found when only [³H] PGE₂ had been added to a sample of gastric juice and averaged 14.5% ± 2.0. Our preliminary results support the possibility that PGE₂ and PGA₂ may be of physiological importance in the regulation of canine gastric secretions.     

Effect of prostaglandin F2α on propulsive activity of the isolated segmental colon of the guinea-pig

The effects of prostaglandin F_2α (PGF_2α) on propulsive activity in segments of isolated colon and on isolated strips of guinea-pig colon were investigated.Using experimental conditions under which spontaneous propulsive activity was negligible, PGF_2α (5×10⁻⁸×1×10⁻⁶M), added to the bathing medium, increased propulsive activity in a concentration dependent manner. This increase of propulsive activity was abolished in the presence of atropine or tetrodotoxin (1×10⁻⁷g/ml).The contractions produced by PGF_2α(5×10⁻⁷ − 1×10⁻⁵M) in isolated longitudinal and circular smooth muscle strips of guinea-pig colon were unaffected in the presence of atropine or tetrodotoxin (1×10⁻⁷g/ml).From these results it is concluded that under the conditions employed in this study propulsive activity stimulated by PGF_2α may depend on the contractions of both muscle layers and stimulation of the peristalic reflex.     

Nuclear and cytoplasmic receptors for 1,25-dihydroxycholecalciferol in intestinal mucosa

The apparent hormonal form of cholecalciferol, 1,25-dihydroxycholecalciferol (1,25-(OH)₂-CC), was incubated with intestinal mucosa homogenates and whole intestinal tissue, $\text{in}\text{vitro}$. After 40–70 min, 1,25-(OH)₂-CC was specifically associated with the nuclear chromatin fraction. This sterol remains bound to the cytosol fraction at 0°C and a dramatic movement to the nuclear chromatin occurs at 37°C indicating that the subcellular localization of the sterol is temperature dependent. Isolated intestinal cytosol, previously incubated with 1,25-(OH)₂-CC, is required for transportation of the hormone to the intestinal chromatin fraction; cytosol fractions from other tissues are ineffective mediators of this sterol migration. It is concluded that the intestinal cytosol contains a specific receptor that functions to transport 1,25-(OH)₂-CC to the nucleus, its probable site of action.     

A microassay of O2 concentration based on oxygen-induced chemiluminescence in anaerobic lipoxygenase reactions

The preparation of chemiluminescence probes for assaying O₂ concentrations in microsamples is described. This probe is based on an anerobic lipoxygenase-linoleate system continously generating reactive intermediates which in a spontaneous reaction with added O₂ yield an excited species. The resulting chemiluminescence signals are highly reproducible upon repeated sample application and unaffected by even large variations in the contents of lipoxygenase-1 and linoleic acid. The linear assay range is between 0.25 and 25 nmol of O₂. The assay system described is stable for 90±10 min. irrespective of the number of samples added, and the probe can be regenerated thereafter by adding linoleic acid.     

Binding of [3H]triamcinolone acetonide-receptor complexes to chromatin from the B-cell leukemia line,BCL1

The binding characteristics of partially purified glucocorticoid receptor complexes from hormone sensitive, non-differentiating BCL₁ cells to sequentially deproteinized BCL₁ chromatin-cellulose was investigated. [³H]Triamcinolone acetonide (TA)-receptor complexes were purified (approx. 30-fold) from DEAF-cellulose columns by salt elution which allowed receptor activation only in the absence of molybdate. Addition of 10 mM molybdate completely blocked salt activation. The binding pattern of the activated [³H]TA-receptor complexes to chromatin-cellulose extracted with 0–8 M guanidine hydrochloride revealed three regions of increased binding activity (acceptor sites), at 2, 5 and 7 M guanidine hydrochloride. Acceptor site binding was markedly reduced for chromatin extracted with 3, 6 and 8 M guanidine hydrochloride. Non-activated receptor complexes demonstrated very low binding to deproteinized chromatin. It was also shown that chromatin binding required glucocortical receptors and that free ligand or ligand bound to other proteins did not bind significantly to chromatin. In addition, binding of [³H]TA-receptor complexes to partially deproteinized chromatin was competable by unlabeled TA-receptor complexes. Scatchard analysis demonstrated that chromatin from non-differentiating BCL₁ cells possesses multiple, high-affinity binding sites which differ in their affinity for the glucocorticoid receptor. Partially deproteinized chromatin from lipopolysaccharide-stimulated BCL₁ cells demonstrated a different pattern of receptor binding, i.e., receptor binding was significantly greater to chromatin previously extracted with 6–8 M guanidine hydrochloride. These results suggest that differentiation alters the state of chromatin and the interaction of non-histone protein/DNA acceptor sites with glucocorticoid receptors. These alterations may play a role in the acquisition of hormone resistance.     

Aggregation of mono- and dinucleosomes into chromatin-like fibers

Three classes of chicken erythrocyte chromatin particles differing in their content of lysine-rich histones and/or spacer DNA have been studied in order to determine their ability to aggregate into complexes resembling those observed in native chromatin. The complexes have been obtained in the presence of MgCl₂ and NaCl and studied by electron microscopy. Mononucleosomes, containing spacer DNA and histones H1 and H5, give rise to thick (about 70 nm) ellipsoidal particles in the presence of 0.5 mM MgCl₂. These particles are disrupted by the addition of small amounts of NaCl (5–20 mM). On the other hand in 0.5 mM MgCl₂ dinucleosomes give rise to regular fibrous complexes of about 40 nm in diameter which are very similar to native chromatin fibers. These complexes are much more stable when NaCl is added. We conclude that for the stability of nucleosomal aggregates, similar to native chromatin fibers, a continuity of DNA structure is not required, but the presence of divalent cations, spacer DNA and lysine-rich histones is essential.     

Ribonucleic Acid Synthesis by Cucumber Chromatin: Developmental and Hormone-induced Changes

When intact etiolated 2-day cucumber (Cucumis sativus) embryos were treated with indoleacetic acid (IAA), gibberellin A₇ (GA₇), or kinetin, chromatin derived from the embryonic axes exhibited an increased capacity to support RNA synthesis in either the presence or the absence of bacterial RNA polymerase. An IAA effect on cucumber RNA polymerase activity was evident after 4 hours of hormone treatment; the IAA effect on DNA template activity (bacterial RNA polymerase added) occurred after longer treatments (12 hours). GA₇ also promoted template activity, but again only after a prior stimulation of endogenous chromatin activity. After 12 hours of kinetin treatment, both endogenous chromatin and DNA template activities were substantially above control values, but longer kinetin treatments caused these activities to decline in magnitude. When chromatin was prepared from hypocotyl segments that were floated on a GA₇ solution, a GA-induced increase in endogenous chromatin activity occurred, but only if cotyledon tissue was left attached to the segments during the period of hormone treatment.     

The structure of Sipunculus nudus erythrocyte chromatin

The structure of the Sipunculus erythrocyte chromatin has been characterized by electron microscopy and nuclease digestion (staphylococcal nuclease and pancreatic nuclease). Contrary to previous results [1], we were able to isolate and characterize a histone H_2B in sipunculid nuclei. Though the histones H_2A and H_2B were markedly different from their vertebrate homologues, the subunit structure of the chromatin is the same. But the length of the repeat unit of DNA in the chromatin, is 177 ± 5 bp for the sipunculid erythrocyte nuclei, close to that reported for the chromatin of some lower eukaryotes.     

Characterization of mouse Taper liver tumor hepatoma chromatin autodigestion and the release of euchromatic segments.

Glycerol density gradient analysis of unsheared mouse Taper liver tumor hepatoma chromatin revealed that the nonpelletable (~20%) chromatin sedimented with a fairly uniform size distribution. Shearing of the chromatin by autodigestion in the presence of added Mg²⁺ and Ca²⁺ resulted in the release of chromatin segments (from the originally pelletable chromatin) with sedimentation chracteristics similar to those of the original nonpelletable chromatin. However, shearing of the chromatin by autodigestion in the absence of added Mg²⁺ and Ca²⁺ or by digestion with added micrococcal nuclease or by physical shearing (Virtis homogenization) resulted in the release of chromatin segments (from the originally pelletable chromatin) which did not display the same sedimentation characteristics. Divalent cation concentration, pH, and incubation temperature were all found to be important factors affecting the final results. Satellite DNA content analyses revealed that the chromatin segments released from the (initially) pelletable chromatin during the early stages of autodigestion (in the presence of added Mg²⁺ and Ca²⁺) were satellite DNA-deficient; at later times during the autodigestion, it appeared that satellite DNA-enriched segments were released from the (initially) pelletable chromatin. A model is proposed which attempts to explain the kinetics of autodigestion.     

Preparation of metaphase chromatin of Physarumpolycephalum without the loss of repressed RNA synthesis

A novel method for the preparation of intact chromatin from the slime mold $\text{Physarum}\text{polycephalum>}$ which retains the $\text{in}\text{vivo}$ property of RNA synthesis is described. Preparations from G₂-cells were highly active, while those from metaphase-cells were inactive. The plasmodial cells were disrupted by gentle homogenization on a polyethylene sieve in a neutral isotonic sucrose medium containing Mg⁺⁺, deoxycholate and EGTA, a Ca⁺⁺-chelating agent. The nuclei were lysed in a hypotonic buffer without use of EDTA and chromatin was precipitated by centrifugation after addition of Mg⁺⁺.     

Controls of Benthic Nitrogen Fixation and Primary Production from Nutrient Enrichment of Oligotrophic, Arctic Lakes

We examined controls of benthic dinitrogen (N₂) fixation and primary production in oligotrophic lakes in Arctic Alaska, Toolik Field Station (Arctic Long-Term Ecological Research Site). Primary production in many oligotrophic lakes is limited by nitrogen (N), and benthic processes are important for whole-lake function. Oligotrophic lakes are increasingly susceptible to low-level, non-point source nutrient inputs, yet the effects on benthic processes are not well understood. This study examines the results from a whole-lake fertilization experiment in which N and P were added at a relatively low level (4 times natural loading) in Redfield ratio to a shallow (3 m) and a deep (20 m) oligotrophic lake. The two lakes showed similar responses to fertilization: benthic primary production and respiration (each 50–150 mg C m^?2 day^?1) remained the same, and benthic N₂ fixation declined by a factor of three- to fourfold by the second year of treatment (from ~0.35 to 0.1 mg N m^?2 day^?1). This showed that the response of benthic N₂ fixation was de-coupled from the nutrient limitation status of benthic primary producers and raised questions about the mechanisms, which were examined in separate laboratory experiments. Bioassay experiments in intact cores also showed no response of benthic primary production to added N and P, but contrasted with the whole-lake experiment in that N₂ fixation did not respond to added N, either alone or in conjunction with P. This inconsistency was likely a result of nitrogenase activity of existing N₂ fixers during the relative short duration (9 days) of the bioassay experiment. N₂ fixation showed a positive saturating response when light was increased in the laboratory, but was not statistically related to ambient light level in the field, leading us to conclude that light limitation of the benthos from increasing water-column production was not important. Thus, increased N availability in the sediments through direct uptake likely caused a reduction in N₂ fixation. These results show the capacity of the benthos in oligotrophic systems to buffer the whole-system response to nutrient addition by the apparent ability for significant nutrient uptake and the rapid decline in N₂ fixation in response to added nutrients. Reduced benthic N₂ fixation may be an early indicator of a eutrophication response of lakes which precedes the transition from benthic to water-column-dominated systems.     

31P-NMR spectrum of phosphocreatine: Deuterium-induced splitting of the signal

It has been found in experiments with high resolution ³¹P-NMR spectroscopy (200 MHz) that the phosphocreatine peak is splitted into two different peaks in the mixtures of H₂O and D₂O and is single but with different chemical shifts in pure H₂O and D₂O. This phenomenon is explained by substitution of protons of guanidino group in phosphocreatine by deuterium. The effect of splitting disappeared at extreme pH values (>8.5 or <4.0) and at temperatures higher than 45°C due to accelerated proton-deuterium exchange. Creatine kinase added to phosphocreatine solution also lowered its temperature of peaks' collapse by 5°–10°C. A saturation (spin) transfer method was used to show that the phosphoryl group transfer to ADP in creatine kinase active center is slower with deuterium-substituted phosphocreatine than with H-phosphocreatine. The data are taken to show the importance of the proton transfer step in the creatine kinase reaction mechanism and acceleration of phosphocreatine proton-deuterium exchange by creatine kinase.     

Characterization of Chromatin Subfractions Enriched in RNA from Lactating Mammary Gland of the Rat

Mammary gland chromatin from lactating rats was fractionated according to the Mg²⁺-solubility method after mild digestion with DNase II. The chromatin properties were compared between the Mg²⁺-soluble (S₂) and the Mg²⁺-insoluble (P₂) fractions. The weight ratio of RNA to DNA was much greater in the S₂ fraction than in the P₂ fraction (S₂, 0.92; P₂, 0.03). The DNA repeat length of the nucleosome was very similar between the two fractions (S₂, 193 ± 7; P₂, 1% ± 8 nucleotide pairs). A significant difference was seen in electrophoretic pattern of H1 histone between the two fractions in the acid-urea gel system. Nonhistone proteins with molecular weights higher than about 40,000 dalton were found to be enriched in the S₂ fraction.