Reactivation of Cytoplasmic Streaming in a Tonoplast-Permeabilized Cell Model of Acetabularia

To find whether cytoplasmic streaming in Acetabularia is controlledby Ca²⁺, a tonoplast-permeabilized cell model was prepared usinga vacuolar perfusion technique. The cytoplasmic streaming remainedalmost normal after perfusion with EGTA medium (10 mM EGTA,40 mM PIPES, 5mM MgCl₂ and 800 mM sorbitol, pH 6.9), but stoppedwithin 10 min when saponin medium (EGTA medium plus 50 µg/mlsaponin, 50 µg/ml hexokinase and 5 mM glucose) was perfused.This model system was reactivated with a solution containing0.5 mM ATP and different concentrations of Ca²⁺ (reactivationmedium). With the reactivation medium at pCa 6–5, theresumed streaming lasted for about 10 min before the cytoplasmaggregated. At pCa 4–3, the streaming was observed onlyfor a few minutes because the cytoplasm aggregated quickly.At pCa 7, no reactivated movement was observed. Reactivationwas not induced in an ATP- or Mg²⁺-deficient medium even inthe presence of an adequate concentration of Ca²⁺, and was inhibitedby 50 µg/ml cytochalasin B or 1 mM N-ethylmaleimide. We concluded from these observations that the cytoplasmic streamingin Acetabularia is very likely to be driven by the actomyosinsystem in the presence of Mg-ATP and Ca²⁺ at pCa 6–5. (Received October 31, 1984; Accepted April 1, 1985)     

Involvement of protein sulfhydryls in the trigger reaction of rhodotorucine A, a farnesyl peptide mating pheromone of Rhodosporidium toruloides.

The involvement of protein sulfhydryls for the signaling of rhodotorucine A, a mating pheromone produced by mating type A cells of Rhodosporidium toruloides, was investigated by the use of sulfhydryl compounds. The sulfhydryl-blocking reagent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB; Ellman's reagent) strongly inhibited both the biological effect of the pheromone on the recipient cell and the hydrolysis of the pheromone, which is catalyzed by the mating type-specific surface endopeptidase of the recipient cell. Conversely, the two reactions were markedly enhanced by the presence of the reducing reagent dithiothreitol. The inhibitory effect of DTNB on the pheromone response of the recipient cell was specific to an initial stage of the differentiation; once it had initiated, the reagent had no effect on its progression. The results suggested that dithiothreitol enhances and DTNB impairs the efficiency with which the pheromone triggers sexual d differentiation. The reaction of DTNB with cellular protein sulfhydryls was highly restricted to those at the exterior surface of the membrane due to the impermeability of the reagent through the membrane. Phosphorylation of endogenous proteins, which is modulated by the pheromone added to an in vitro phosphorylation system, was also blocked by DTNB. The results showed that sulfhydryl groups are involved in the pheromone hydrolysis by the surface endopeptidase of the recipient cell and that pheromone metabolism is indispensable for the signaling reaction. We suggest that the modulation of protein phosphorylation of membrane proteins by the pheromone is an initial transmembrane response coupled to pheromone metabolism.     

Effect of various catecholamine antagonists on prolactin secretion in conscious male rabbits

To clarify physiological roles of catecholaminergic systems in the control of rabbit prolactin (PRL) release, the effect of various catecholamine receptor antagonists on plasma PRL levels was examined in conscious, freely moving male rabbits. An intravenous (iv) injection of yohimbin (2.5 mg/kg body wt), an alpha 2-adrenoreceptor antagonist, but not prazosin (2 mg/kg body wt), an alpha 1-adrenergic receptor antagonist, resulted in a significant elevation of plasma PRL. Conversely, propranolol (2.5 mg/kg body wt, iv), a nonselective beta-adrenoreceptor antagonist, and metoprolol (2.6 mg/kg body wt, iv), a beta 1-adrenergic antagonist, slightly but significantly suppressed basal levels of plasma PRL. On the other hand, haloperidol (0.5 mg/kg body wt, iv), pimozide (0.3 mg/kg body wt, iv), sulpiride (5 mg/kg body wt, iv), chlorpromazine (3 mg/kg body wt, iv), and YM-09151-2 (0.2 mg/kg body wt, iv), all dopamine receptor antagonists caused a significant increase in plasma PRL. These results suggest that dopaminergic and alpha 2-adrenergic mechanisms exert a tonic inhibitory role and beta-adrenergic mechanisms, probably beta 1, a tonic stimulatory role in the regulation of PRL release in the rabbit.     

Identification of an Rts1 DNA fragment conferring temperature-dependent instability to vector plasmids

The multiphenotypic drug resistance factor Rts1 expresses a temperature-dependent instability characteristic. This plasmid was digested with the restriction enzyme BamHI. A DNA fragment with a molecular weight of 5.6 MDa (the H fragment) was inserted into plasmid pBR322 (pFK896) or into pSC105 (pYH156) at the BamHI site. These plasmids were unstable at 42 degrees C but stable at 32 degrees C. A restriction-enzyme map of the H fragment was constructed and the instability phenotype (Tdi) was localized to a DNA fragment with 0.5 MDa molecular weight. The temperature-dependent loss of the unstable plasmid pFK896 is abrupt and no gradual plasmid loss of this multicopy recombinant plasmid is observed. The possibility that the Tdi phenotype is due to overgrowth of R- cells was eliminated.     

Changes of aldolase A and B messenger RNA levels in rat liver during azo-dye-induced hepatocarcinogenesis

The expression of aldolase A and B mRNAs during azo-dye-induced carcinogenesis in rat liver was examined. After feeding the dye for 18 weeks, the level of aldolase A mRNA increased to about 11 times that in a normal liver, with the concomitant decrease of aldolase B mRNA level to about 25% of that in a normal liver. These changes did not occur progressively during the carcinogenesis, but occurred as an additional phase after 4 week-feeding of the azo-dye. At this stage, the levels of aldolase A and B mRNAs were about 7 times and 45% of that in a normal liver, respectively. This biphasic pattern in the aldolase isozyme expression in the azo-dye-fed rat liver is discussed together with the kinetic data of the enzyme activity.     

Transformation-defective Rous sarcoma virus mutants with altered p19 of the gag gene and their inhibitory effect on host cell growth.

Mutants (PH2010, PH2011, PH2012) of Rous sarcoma virus which have a growth-inhibitory effect on chicken embryo fibroblasts were isolated from a temperature-sensitive mutant of the Schmidt-Ruppin strain of Rous sarcoma virus (tsNY68). The growth rate of fibroblasts infected with these viruses was about 50 to 60% of that of uninfected fibroblasts. A morphological difference between mutant-infected and uninfected fibroblasts was observed at logarithmic phase but not at stationary phase. Neither the protein p60src nor its associated protein kinase activity was significantly detected by an immunoprecipitation assay in the cells infected with these mutants. Analysis of the unintegrated DNA of the mutant PH2010 showed that a sequence of about 1.4 kilobase pairs at the src gene region is deleted. Further examination of the viral structural proteins in infected cells as well as in virions by immunoprecipitation and peptide mapping revealed that the molecular size of the Pr76 gag protein of the mutant RSV is smaller than that of the mutant tsNY68 because of partial deletion at the p19 gag gene. The peptide maps suggest that the deleted region of the altered p19 of the mutant is near the carboxy terminal of p19. The amount of Prgp92env synthesized in the mutant-infected cells was about fivefold more than that in tsNY68-infected cells.     

Stable and unstable phase of memory in classically conditioned fly, Phormia regina: Effects of nitrogen gas anaesthesia and cycloheximide injection

This study examined the effects of nitrogen anaesthesia and cycloheximide injection on memory of the classically-conditioned fly, Phormia. 1 M NaCl solution was given to each fly as a conditioning stimulus and 0.5 M sucrose solution was the unconditioned stimulus that induced the proboscis extension response. The training period was as short as 2 min and testing was usually carried out 2 hr later. At varying times (0–60 min) between training and testing, flies were anaesthetized with nitrogen gas for 25 sec. When flies were anaesthetized immediately after training the effect of nitrogen gas was the greatest and few flies showed any conditioned response, but the sensitivity of memory to nitrogen gas declined as the interval between training and nitrogen treatment became longer, and such treatment had no effect on memory when the interval was longer than 30 min. The effect on memory of cycloheximide, an inhibitor of peptide bond synthesis, was also investigated. The injection of cycloheximide (0.37 μg) immediately after training diminished the memory, but when given 1 hr after training it had no effect on memory. These results show that the memory in Phormia has two phases, stable and an unstable phase, like long-term and short-term memory in vertebrates.     

Occurrence of beta-chain conformation in poly-gamma-methyl glutamate membranes

Molecular chain conformations of poly-γ-methyl-L -glutamate, poly-γ-methyl-D -glutamate, and poly-γ-methyl-D ,L -glutamate in membranes prepared by using mainly trifluoroacetic acid and formic acid as solvents were investigated by infrared, X-ray diffraction, and optical rotatory dispersion measurements. It was pointed that these polymers exist in the α-helix form in membranes cast from trifluoroacetic acid solutions, but in the β-chain form in membrances swollen in formic acid. The β-chain structure was also observed in crystals precipitated from dilute solutions including formic acid. The formation of the β-chain structure was discussed.