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.     

Angiostrongylus cantonensis: Development following pulmonary arterial transfers into permissive and nonpermissive hosts

The survival, growth, and egg-laying capacity of young adult Angiostrongylus cantonensis, surgically transferred from intracranial sites into pulmonary arteries, were studied. A variety of experimental animals (rats, guinea pigs, mice, and mastomys) were chosen as donor animals and as recipient hosts (rats, guinea pigs, and rabbits). These species were specifically chosen to span the spectrum of host permissiveness relative to worm development in an attempt to understand the mechanisms which underlie species-dependent resistance. Recipient animals were monitored not only for the development of parasites per se but also for antibody production and histopathologic changes. The results indicated that these procedures were technically feasible, with good worm development following intra-rat transfers, as early as 15 days after initial exposure. Studies were performed to analyze the constraints of development both on initial, i.e., prelung and subsequent i.e., postlung development. When worms were obtained from permissive species such as rat or mastomys, transfer into rats resulted in good growth and development; however, worms which developed initially in exposed mice or guinea pigs developed less well in the rat. Conversely, worms which developed initially in permissive host such as the rat, when transferred into a variety of less permissive hosts such as the guinea pig and rabbit, apparently did not survive and caused significant morbidity and mortality within the nonpermissive host. Histopathologic evaluation revealed a strong eosinophilic perivascular and peribronchiolar infiltration as well as granulomatous reactions surrounding the worms in the lungs of recipient guinea pigs and rabbits, changes not observed in the lungs of permissive rat recipients. As reaginic antibody responses were also more prominent in nonpermissive than in permissive animals, it is possible that IgE responses may be more directly related to the phenomenon of morbidity and/or permissiveness than are other aspects of immune response. In support of this contention was the finding of nearly equivalent hemagglutinating antibody production between permissive rats and nonpermissive guinea pigs and rabbits.     

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.     

Cholera enterotoxin production in Vibrio cholerae O1 strains isolated from the environment and from humans in Japan.

Vibrio cholerae O1 strains isolated from various sources in Japan over the years 1977 through 1987 were examined to confirm the presence or absence of the cholera enterotoxin (CT) gene and production of CT and to determine the kappa-phage type. The CT gene was detected in none of 225 isolates from natural waters but was present in all of the 10 isolates from environmental waters implicated in domestic cholera cases, in 64 strains (26.6%) of the 241 isolates from imported seafoods, in 43 strains (95.6%) of the 45 isolates from domestic cholera cases, and in 119 strains (93.7%) of the 127 isolates from imported cholera cases. The results suggest that the CT gene-positive strains of V. cholerae O1 have been imported into Japan through seafoods and/or by travelers. Sporadic cholera cases have resulted in contamination of the surrounding environment, but the CT gene-positive strains may not have persisted in natural waters to serve as a reservoir for epidemic cholera. The commercially available VET-RPLA kit (a latex agglutination kit for immunological detection of CT) detected production of CT in all of the CT gene-positive strains, indicating that there was no silent CT gene in the test strains. There was a strong correlation between the kappa-phage type and the presence or absence of the CT gene, suggesting a significant clonal difference between CT gene-positive and -negative strains. Five CT gene-negative strains isolated from imported cholera cases (travelers with mild diarrhea) induced a considerable amount of fluid accumulation in rabbit and/or suckling mouse intestines, indicating production of an enterotoxic factor(s) other than CT.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.