Amino acid incorporation by nuclear membrane fraction of rat liver.

Nuclear membrane fraction of rat liver is able to incorporate ¹⁴C-leucine into its proteins $\text{in vitro}$. The incorporation of ¹⁴C-leucine into the nuclear membrane fraction was almost completely inhibited by chloramphenicol, but the inhibition by cycloheximide and puromycin was not so remarkable. RNase and DNase were not effective. The incorporation was also inhibited by several reagents known to interfere with energy metabolism. These characteristics of the incorporation of ¹⁴C-leucine by the nuclear membrane fraction are quite similar to those of the incorporation by nuclei isolated from rat liver and mitochondrial fraction, but seem to be different from those of the ordinary protein synthetic system in microsomal fraction. ¹⁴C-Leucine was preferentially incorporated into intrapolypeptide or C-terminal residues but not into N-terminal residues. Acrylamide gel electrophoresis showed that three protein species were mainly labelled. The incorporating activity of the nuclear membrane fraction obtained from regenerating liver 17 h after partial hepatectomy showed 220 % of the control. The possibility that the contaminated mitochondrial fraction might be responsible for the incorporation of ¹⁴C-leucine by the nuclear membrane fraction was ruled out.     

Thermodynamic characterization of cytochrome c at low pH. Observation of the molten globule state and of the cold denaturation process.

Several reports have pointed out the existence of intermediate states (both kinetic and equilibrium intermediate) between the native and the denatured states. The molten globule state, a compact intermediate state in which the secondary structure is formed but the tertiary structure fluctuates considerably, is currently being studied intensively because of its possible implication in the folding process of several proteins. We have examined the thermal stability of horse cytochrome c at low pH between 2.0 and 3.2 and different potassium chloride concentrations by absorbance of the Soret band, far and near-ultraviolet circular dichroism (u.v. c.d.) and tryptophan fluorescence using a multidimensional spectrophotometer. The concentration of potassium chloride ranged from 0 M to 0.5 M. The experimental thermal denaturation curves show that: (1) the helical content of cytochrome c remains stable at higher temperature when the concentration of salt is increased; whereas (2) the extent of ordering of the tertiary structure is weakly dependent on salt concentration; and (3) for cytochrome c, the stabilization of the molten globule state is induced by the binding of anions. Other salts such as NaCl, LiCl, potassium ferricyanide (K3Fe(CN)6) and Na2SO4 may also be used to stabilize the molten globule state. The thermodynamic analysis of the denaturation curves of c.d. at 222 nm and c.d. at 282 nm shows that, whereas a two-state (native and denatured) transition is observed at low-salt concentration, the far and near-u.v. c.d. melting curves of cytochrome c do not coincide with each other at high-salt concentration, and a minimum of three different thermodynamic states (IIb, intermediate or IIc, and denatured) is necessary to achieve a sufficient analysis. The intermediate state (called IIc) is attributed to the molten globule state because of its high secondary structure content and the absence of tertiary structure. Therefore, at low pH, cytochrome c is present in at least four states (native, IIb, IIc and denatured) depending on the salt concentration and temperature. The thermodynamic parameters, i.e. the Gibbs free energy differences (delta G), the enthalpy differences (delta H), the midpoint temperatures (Tm) of the transition (IIb in equilibrium intermediate (IIc in equilibrium denatured) are determined. We also give estimates of the heat capacity differences (delta Cp) from the temperature dependence of the enthalpy differences. The enthalpy change and the heat capacity difference of the IIc in equilibrium denatured transition are non-zero. The number of charges (protons or chloride anions) released upon transitions are determined by analysing the pH and chloride anion concentration dependence of the Gibbs free energy.(ABSTRACT TRUNCATED AT 400 WORDS)     

Change in the rate of organelle movement during progression of the cell cycle inAdiantum protonemata

Summary The rate of organelle movement during progression of the cell cycle in single-celled protonemata of the fernAdiantum capillus-veneris is determined microscopically with a time-lapse video system. Under red light organelle movement is very slow (1.8 m/min) in early G₁ in the apical 100-m region. The rate of organelle movement becomes higher in proportion to distance from the nuclear region, reaching a plateau in the neighborhood of 300 m from the tip. Organelle movement during the progression of G₁ and S phases in the dark does not show a significant difference from that in early G₁ under red light. In M phase, however, organelle movement in the nuclear region slows down a few minutes after nucleolar disappearance and then stops until the beginning of cell plate formation. Organelle movement in the basal region of the protonema slows down, but does not stop, shortly after movement in the nuclear region has ceased. This indicates that a message is sent from the nuclear region to the basal region.     

Simultaneous analyses of phenethylamine, phenylethanolamine, tyramine and octopamine in rat brain using fluorescamine

A fluorometric method for the simultaneous analyses of phenethylamine, phenylethanolamine, tyramine and octopamine has been developed. The method involves ion-exchange chromatography, derivatization with fluorescamine, solvent extraction and then separation by thin-layer chromatography. The fluorescent spots are then quantitated by scanning. The detection limits of this method are about 10 pmoles for phenethylamine, phenylethanolamine and tyramine, and 20 pmoles for octopamine. The method was used for simultaneous analyses of putative neurotransmitter amines in whole rat brain.     

Isolation and characterization of androgen-dependent non-histone chromosomal protein from dorsolateral prostate of rats

Rat prostate contains a unique androgen-dependent non-histone protein (Matuo et al. (1)). The non-histone protein was isolated in homogeneous form by extraction of nuclei from the dorsolateral prostate with 0.35 M NaCl in the presence of 1 mM PMSF and chromatography on a CM-Sepharose column. The final fraction was greater than 98% pure as judged by electrophoreses in SDS- and acid/urea-gels. The purified protein had a molecular weight of approximately 20,000, and an isoelectric point of approximately 11.5. Its absorption peak was at 276 nm and A(1%, 276nm)=9.3. The protein is characterized by the absence of cysteine, histidine and tryptophan, and by the high content of methionine, tyrosine and phenylalanine.     

Superoxide anion reacts with enzyme intermediate in the bacterial luciferase reaction competitive with intramolecular electron transfer

Addition of KO₂ in dimethyl sulfoxide (DMSO) to the in vitro bacterial luciferase reaction subsequent to its initiation resulted in a biphasic decay of light emission. The first and more rapid phase is attributed to quenching by DMSO. With DMSO alone the continuing decay is kinetically the same as in a control reaction. With KO₂ added the second decay phase is more rapid and dependent on the KO₂ concentration. The enhanced decay is attributed to superoxide anion generated from KO₂ reacting without light emission with an enzyme peroxy intermediate, breaking down of the peroxide bond through intermolecular electron transfer from the superoxide anion, in competiton with an intramolecular electron transfer from the N(5) position of the flavin ring, which normally leads to the production of the excited luciferase-dihydroflavin-4a-hydroxide. © 1997 John Wiley & Sons, Ltd.     

Cytological, genetic and evolutionary functions of chiasmata based on chiasma graph analysis.

The nature of the chiasma as a cytological parameter for analysing cross-over was reexamined quantitatively by an improved chiasma graph method. It was reconfirmed in Mus platythrix (n =13) that interstitial chiasmata at diakinesis are distributed randomly and almost uniformly along bivalents except for the centromere and telomere regions. The size of these chiasma blank regions was consistently 0.8% of the total length of haploid autosomes in all chromosomes. There was a minimum value of chiasma interference distance between two adjacent chiasmata, which was constantly 1.8% in all chromosomes. The chiasma frequency at diakinesis was 20.1+/-2. 0 by the conventional method including terminal chiasmata. However, the primed in situ labeling technique revealed that terminal chiasmata were mostly telomere-telomere associations. From these data and also from recent molecular data we concluded that the terminal chiasma is cytologically functional for ensuring the normal disjunction of bivalents at anaphase I, but genetically non-functional for shuffling genes. The chiasma frequency excluding terminal chiasmata was 14.6+/-1.8. Reexamination of the chiasma frequency of 106 animal species revealed that the chiasma frequency increased linearly in proportion to the haploid chromosome number in spite of remarkable difference in their genome size. The increase in chiasma frequency would be evolution-adaptive, because gene shuffling is expected to be accelerated in species with high chromosome numbers.     

Effect of suplatast tosilate (IPD-1151T) on cytokine production by allergen-specific human Th1 and Th2 cell lines.

Suplatast tosilate (IPD-1151T) is an antiallergic agent that suppresses airway eosinophil infiltration in asthma. We investigated the effects of IPD-1151T on proliferative response and cytokine production by human antigen-specific T cell lines. Purified protein derivatives (PPD)-specific T helper 1 (Th1) cell lines and Dermatophagoides farinae (Der f)-specific T helper 2 (Th2) cell lines were established from patients with asthma sensitized with house dust mite. Stimulation of PPD-specific and Der f-specific T cell lines with relevant antigens resulted in production of mostly interferon (IFN)-gamma and of interleukin (IL)-4 and IL-5, respectively. IPD-1151T did not inhibit the proliferative responses of either the Th1 or Th2 cell line to antigens. Although IPD-1151T did not inhibit IFN-gamma production by PPD-specific Th1 cell lines, it did inhibit IL-4 and IL-5 production by antigen-stimulated Der f-specific Th2 cell lines in a dose-dependent manner. IPD-1151T directly inhibited cytokine production by Der f-specific Th2 cell lines stimulated with immobilized anti-CD3 antibodies. Although IPD-1151T did not inhibit the clonal expansion of memory T cells among PBMCs into PPD-specific Th1 and Th2 cell lines, it did inhibit IL-4 and IL-5 production by Der f-specific Th2 cell lines but not IFN-gamma production by PPD-specific Th1 cell lines. These results suggest that IPD-1151T selectively inhibits Th2-type cytokine production.