Assembly of nucleosomes: the reaction involving X. laevis nucleoplasmin

We analyze the nucleosome core assembly reaction which is mediated in vitro by a protein previously purified from Xenopus laevis eggs, now named nucleoplasmin in reference to its occurrence in the soluble phase of the nucleus of a wide range of vertebrate cell types. Nucleoplasmin is present in solution as a pentamer. We use nuclease digestion analysis to show that the protein assembles bona fide nucleosome cores in vitro from purified histones and DNA. Nucleoplasmin itself binds neither to DNA nor to the nucleoprotein particles which it assembles in vitro. However, it interacts with histones in vitro in such a way that histones no longer adhere to negatively charged surfaces. We have found no evidence for sterically specific interactions with particular histones. The initial rate of the nucleosome core assembly reaction mediated by purified nucleoplasmin in vitro is essentially identical with the rate of the nucleosome assembly reaction which occurs in the cell-free extracts of Xenopus eggs from which nucleoplasmin was purified. This rate is sufficient to account for the rate of nucleosome assembly required during the early development of Xenopus embryos.     

Both induction and activation of the branched-chain 2-oxo acid dehydrogenase complex in primary-cultured rat hepatocytes by clofibrate

Clofibrate administration to rats caused both the activation and induction of the branched-chain 2-oxo acid dehydrogenase complex in the liver; the former phenomenon occurred within the first 6 h after clofibrate administration whereas the latter occurred after 12 h. Essentially the same results were obtained with primary cultures of rat hepatocytes in the presence of 0.5 mM clofibrate, though about three-fourths of the enzyme complex in control cells (without clofibrate addition) was inactivated during a culture for 44 h, with little reduction of the enzyme amount. This was also confirmed by immunotitration analysis with antibodies raised against the purified decarboxylase and transacylase components of the enzyme complex. On the other hand, the activity of dihydrolipoamide dehydrogenase (a constituent of the complex) was little affected by clofibrate administration. The half lives of the decarboxylase and transacylase components in the primary cultures were estimated to be in the range of 22-26 h, and were unchanged in the presence of clofibrate, when determined with the use of cycloheximide and by a pulse-chase experiment. On the contrary, the rates of synthesis of these two enzyme components had increased to about 1.9-fold after 32 h cultivation in the presence of clofibrate. Thus, the increase in the synthesis of both the components resulted in induction of the complex.     

Demonstration of platelet activating factor receptor in guinea pig kidney.

Distribution of platelet activating factor (PAF) receptor was examined in the guinea pig kidney. Northern blot analysis showed a single band electrophoresed just below the 28S rRNA, and the mRNA was richest in the cortex with lesser amounts in the outer and then inner medulla. Scatchard analysis of membrane fraction using [3H]WEB 2086, a specific PAF receptor antagonist, revealed a single binding site with Bmax of 522, 228, 58 fmol/mg protein for the cortex, outer medulla and inner medulla, respectively. Kd values were in the same order of magnitude (10(-8) M). These results indicate the presence of a single class of PAF receptor in the guinea pig kidney which is most abundant in the cortex.     

Solution structure of human growth hormone-releasing factor fragment (1-29) by CD: characteristic conformational change on phospholipid membrane

The conformations of synthetic human growth hormone-releasing factor fragment (1-29) in the presence and the absence of 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol liposome as well as in aqueous 2,2,2-trifluoroethanol solution were investigated by CD spectroscopy. The secondary structure of the peptide in each solution was analyzed by two methods. Both results show that the peptide has an unordered structure in the aqueous solution, whereas it folds into helical structure in the aqueous alcohol and in the phospholipid solution. In addition, although the peptide exists as almost complete helix in the 50 vol% aqueous alcohol (80-90% helicity), it does not reach full helicity even in the solution containing excess amount of phospholipid liposome (maximum 65-70% helicity). The conformational difference is explained by the characteristic amphipathy of the peptide, i.e., the necessity to twist the separated amphipathic helical parts in the interaction with the phospholipid membrane probably makes the helicity of the peptide decrease.     

Suppression of the degradation of recombinant human apolipoprotein E by a protease inhibitor obtained from fetal bovine serum in serum-free culture

The recombinant human apolipoprotein E (Apo-E) produced by Chinese hamster ovary cells (CHO-322 cells) in serum free culture was degraded to 24K and 23K fragments that contained N-terminal amino acid. The degradation site of Apo-E to 24K fragment was between Arg180 and Leu181 and the C-terminal amino acid of 23K fragment was Gly169. In fetal bovine serum (FBS)-containing culture, the degradation was inhibited. However, in calf serum (CS) the inhibitory activity was not detected. Thus, we attempted the purification of the factor with this inhibitory activity from FBS. A protease inhibitor was purified to give a single peak from FBS by ammonium sulfate precipitation and combination of several column chromatographies. When this FBS-derived protease inhibitor (FBS-d-PI) was added to serum-free culture of CHO-322 cells, degradation of recombinant Apo-E to the 24K and 23K fragments was dose-dependently suppressed and accumulation of intact Apo-E in culture supernatant was observed. FBS-d-PI was found to be a glycoprotein with relative molecular size of 75K daltons under reducing condition, and 85K daltons under nonreducing condition by SDS-PAGE. A complex of FBS-d-PI and a cellular protease was also detected in culture supernatant by western blot analysis using mouse monoclonal antibodies against FBS-d-PI.     

A kinetic analysis of cell disruption by bead mill. The influence of bead loading, bead size and agitator speed.

The influence of operating parameters such as bead loading, peripheral velocity and bead size on the kinetic behavior of cell disruption in a bead mill was investigated. The bead mill was equipped with a single rotating disc and operated batchwise. Analysis of the data showed that the frequency of bead collision may be correlated to the observed first-order process, applying a new concept called effective disruption volume. It was found that the first-order rate constant was proportional to the square of bead loading within the other experimental conditions examined and increased with the decrease in bead diameter. A new disruption kinetics was proposed, explaining all the observed data in terms of the frequency of bead collision and the concept of effective disruption volume. Although other types of microorganisms were not examined, the concept may well be extended to various kinds of cells.     

Respiratory and circulatory activities in carotid body-resected humans.

The respiratory and circulatory activities of patients who underwent carotid body resection (CBR) more than two decades ago were reviewed. No significant ventilatory response to continuous hypoxia was observed. However, in response to stimulation of peripheral chemoreceptors, transient hyperventilation occurred before hypoxemic blood arrived at the central nervous system (single-breath test), which indicated the presence of weak peripheral chemosensitivity. Because of this slight residual peripheral chemosensitivity, which was found shortly after the operation and apparently remained more or less unchanged for greater than 20 years, peripheral chemoreceptor activity, which has been reported in other animal species, does not seem to have returned. Delayed hypoxic hyperventilation reported in dogs and cats with CBR was not observed. Hypoxia significantly depressed the ventilatory response to CO2, but the delayed ventilatory depression with time that has been demonstrated in normal subjects did not occur. In our circulatory studies, hypoxia augmented the heart rate and slightly depressed the stroke volume and total peripheral resistance in the systemic circulation but induced no appreciable changes in arterial blood pressure or cardiac output. We used these results to partition the relative contributions to the overall circulatory response of carotid body stimulation, pulmonary inflation, and other modifying influences. From these calculations, it was inferred that the carotid body reflex plays a dominant role in vascular activities whereas the pulmonary inflation reflex dominates in cardiac activities in humans.     

Parathyroid hormone-related protein is a possible autocrine growth inhibitor for lymphocytes

Adult T-cell leukemia (ATL)-related cells have the ability to produce a newly-isolated calcium-regulating protein, parathyroid hormone-related protein (PTHrP). The present study revealed that lectin-stimulated normal lymphocytes produce immunoreactive (IR)-PTHrP. When the T-cell-enriched fraction was purified from normal lymphocytes and then treated with lectin, a similar amount of IR-PTHrP was detected, suggesting that IR-PTHrP is an actual product of T-lymphocytes. A biologically active fragment of PTHrP, PTHrP(1-34), suppressed DNA synthesis in lectin-stimulated lymphocytes at concentrations greater than 50 pg/mL; the same concentration range of IR-PTHrP detected in the cultured media of lectin-stimulated lymphocytes. Therefore, it is reasonable to postulate that PTHrP is a cytokine inhibiting the cellular growth of normal lymphocytes.