The levels of ecdysteroids in uninjured and leg-autotomized nymphs of Blattella germanica (L.)

The levels of ecdysteroids in control and leg-autotomized first-instar nymphs of Blattella germanica were determined by radioimmunoassay from hatching to the time of the first ecdysis. Uninjured nymphs showed a distinct release of ecdysteroids half-way through the stadium, and this resulted in the commencement of the moult cycle which formed the cuticle of the second instar. Cockroaches which had legs autotomized at 48 h after hatching (i.e. before the control ecydsteroid release) had their instar duration increased by that time period. Releases of ecdysteroids and events of the moulting cycle were also postponed by the 48 h period. The titre of ecdysteroids in injured animals was double that of controls. Nymphs were also autotomized at 96 h (i.e. after the normal release of ecdysteroids) but no changes in instar duration, ecdysteroid releases, or events of the moult cycle were recorded. The effects of injury, prothoracicotropic hormone activity and ecdysteroid release are discussed.     

Effects of decapitation and ovariectomy on the regulation of juvenile hormone synthesis in the cockroach, Diploptera punctata

Corpora allata from Diploptera punctata females at adult ecdysis or at the end of the last-larval stadium, when implanted into decapitated females, underwent a cycle of juvenile hormone synthesis similar in timing and magnitude to that of glands implanted into control animals which had been starved and allatectomized. Starvation did not alter the cycle in rates of juvenile hormone synthesis of sham-operated animals.Decapitation of ovariectomized animals resulted in no cycle in rates of juvenile hormone synthesis by implanted adult corpora allata; however, implantation of an ovary along with the corpora allata into decapitated, ovariectomized hosts resulted in a cycle of juvenile hormone synthesis. In control animals, which retained their heads but were starved and allatectomized as well as ovariectomized, the implanted corpora allata showed a cycle of juvenile hormone synthesis only when implanted with an ovary. The maximal rates of juvenile hormone synthesis by the corpora allata in both experimental and control conditions were lower than normal, likely due to the repeated trauma of surgery. However, at no time from eclosion to the end of the first gonotrophic period was the brain necessary for the cyclic response of the corpora allata to the presence of the ovary.     

The effect of various inhibitors of DNA synthesis on the repair of DNA photoproducts

The effect on DNA repair of several inhibitors of DNA synthesis has been investigated in CHO cells. Three assays were employed following ultraviolet irradiation of G₁ cells: unscheduled DNA synthesis, removal of antibody binding sites and alkaline elution. Cytosine arabinoside and aphidicolin were found to reduce unscheduled DNA synthesis in a dose-dependent manner without affecting the removal of antibody-binding sites. Strand rejoining was also inhibited. These results are consistent with the hypothesis that inhibition is due to premature chain termination during repair synthesis some time after excision of the lesion. Conversely, inhibition of unscheduled DNA synthesis by novobiocin is paralleled by inhibition of excision of the lesion. However, no inhibition of incision was apparent. Since nalidixic acid, an inhibitor of topoisomerase II, did not inhibit excision, it is unlikely that the primary site of action of novobiocin is this topoisomerase. The possibility that a second topoisomerase and/or a polymerase are affected is discussed in the light of previously published data.     

Acceptor activity, isoacceptor profiles and function in protein synthesis of transfer RNAs from regenerating skeletal muscle

Transfer RNAs have been prepared from control and regenerating rat skeletal muscle. The yield of tRNA is highest during the early stages of the regeneration process (5 and 8 days following the induction of regeneration) and decreases to near control values thereafter. The amino acid acceptor activity (extent of aminoacylation) of tRNA from regenerating muscle was also found to be higher for some amino acids than the activity of control tRNA, and the maximum increase in activity was observed between 5 and 8 days following the initiation of regeneration with a decrease to control levels through 15 and 30 days. The isoacceptor pattern, determined by RPC-5 chromatography, for methionyl-tRNAs from control muscle and 5-day regenerating muscle were essentially indistinguishable, while a minor peak of prolyl-tRNA was observed in the population from 5-, 8- and 15-day regenerates which was apparently absent from the control tRNA. Lysyl-tRNAs from control muscle contain two major isoacceptors while a third isoacceptor is observed in the tRNA preparations from 5-, 8- and 15-day regenerating muscle. The relative amount of this third isoacceptor is highest in the 8-day population and decreases in amount in tRNAs from 15- and 30-day regenerates. Control muscle also contains two major glutamyl-tRNA species while a third isoacceptor can be detected in regenerates. The relative amount of this species increases during the early course of the regeneration process but is present at near control levels by 30 days following Marcaine injection. Cell-free protein synthesis using muscle polyribosomes showed that tRNAs from regenerating muscle were more effective in stimulating [³⁵S]methionine incorporation than tRNAs from control muscle.     

Effects of temperature changes on thymidine kinase in heat- and cold-sensitive cell-cycle mutants and ‘wild-type’ murine P-815 cells

Two heat-sensitive (arrested in G1 at 39.5°C) and two cold-sensitive (arrested in G1 at 33°C) clonal cell-cycle mutants that had been isolated from the same clone (K 21), of the murine mastocytoma P-815 cell line, were tested for thymidine kinase (EC 2.7.1.21) activity. After shift of mutant cells to the nonpermissive temperature, thymidine kinase activity decreased, and minimal levels (i.e., less than 3% of those observed for ‘wild-type’ K 21 cells at the respective temperature) were attained within 16 h in heat-sensitive and after 3–4 days in cold-sensitive mutants, which is in good agreement with kinetics of accumulation of heat-sensitive and cold-sensitive cells in G1 phase. After return of arrested mutant cells to the permissive temperature, thymidine kinase of heat-sensitive cells increased rapidly and in parallel with entry of cells into the S phase. In cultures of cold-sensitive cells, however, initiation of DNA synthesis preceded the increase of thymidine kinase activity by approx. one cell-cycle time. Thymidine kinase activities in revertants of the heat-sensitive and cold-sensitive mutants were similar to those of ‘wild-type’ cells. In ‘wild-type’ K 21 cells incubated at 39.5°C, thymidine kinase activity was approx. 30% of that at 33°C. This difference is attributable, at least in part, to a higher rate of inactivation of the enzyme at 39.5°C, as determined in cultures incubated with cycloheximide. The rapid increase of thymidine kinase activity that occurred after shift of K 21 cells and of arrested heat-sensitive mutant cells from 39.5°C to 33°C was inhibited by actinomycin D and cycloheximide.     

Requirements for antiribosomal activity of pokeweed antiviral protein

It has been known for some time that pokeweed antiviral protein acts by enzymatically inhibiting protein synthesis on eucaryotic ribosome systems. The site of this action is known to be the ribosome itself. In this paper we show that the pokeweed antiviral protein reaction against ribosomes is a strong function of salt concentrations, where 160 mM K⁺ and 3 mM Mg²⁺ retards the reaction, while 20 mM K⁺ and 2 mM Mg²⁺ allows maximum reaction rate. It is also shown, however, that an unidentified protein in the postribosomal supernatant solution, together with ATP, allows the ribosome to be attacked even in the presence of high salt. Kinetic analysis of the antiviral protein reaction has been carried out under both sets of conditions, and reveals that the turnover number for the enzyme is about 300–400 mol/mol per min. in each case. The K_m for ribosomes is 1 μM in the presence of low salt and 0.2 μM at higher salt in the presence of postribosomal supernatant factors plus ATP. The antiviral protein reaction is also shown to be pH dependent and is controlled by a residue with pK_a value of approx. 7.0, apparently a histidine. Stoichiometric reaction of the enzyme with iodoacetamide results in a significant loss of antiribosomal activity.     

Translation of poly(U) on ribosomes from Streptomyces aureofaciens

Slowly cooled cells of Streptomyces aureofaciens contained mainly tight-couple ribosomes. Maximum rate of polyphenylalanine synthesis on ribosomes of S. aureofaciens was observed at 40°C, while cultures grew optimally at 28°C. Ribosomes of S. aureofaciens differed from those of E. coli in the amount of poly(U) required for maximum synthetic activity. The polyphenylalanine-synthesizing activity of E. coli ribosomes was about 3-times higher than that of S. aureofaciens ribosomes. The addition of protein S1 of E. coli or the homologous protein from S. aureofaciens had no stimulatory effect on the translation of poly(U). In order to localize alteration(s) of S. aureofaciens ribosomes in the elongation step of polypeptide synthesis we developed an in vitro system derived from purified elongation factors and ribosomal subunits. The enzymatic binding of Phe-tRNA to ribosomes of S. aureofaciens was significantly lower than the binding to ribosomes of E. coli. This alteration was mainly connected with the function of S. aureofaciens 50 S subunits. These subunits were not deficient in their ability to associate with 30 S subunits or with protein SL5 which is homologous to L7/L12 of E. coli.     

Metabolism of parenterally administered zinc and cadmium in livers of newborn rats

The interaction of injected zinc and cadmium with metallothionein was investigated in newborn rats. Tissues of 5-day-old rats were removed 24 h after a single injection (Sc) of saline or zinc (20 mg/kg, body wt.) or cadmium (1 mg/kg, body wt.) with 2.5 μCi of ⁶⁵Zn or ¹⁰⁹Cd or 5 μCi of [³⁵S]cysteine. Injection of zinc resulted in a 75% increase in the hepatic zinc concentration with a concomitant elevation of metallothionein (P < 0.001), zinc in metallothionein increased by 45% (P < 0.05); [³⁵S]cysteine incorporation indicated the induced synthesis of metallothionein. Injection of cadmium did not alter either metallothionein or zinc levels in liver, but cadmium in cytosol was preferentially bound to metallothionein. Neither treatment altered hepatic copper metabolism and copper in metallothionein, nor renal zinc and metallothionein levels. These data indicate that zinc injection can elevate hepatic zinc levels and induce metallothionein synthesis in newborn rats despite high basal levels; cadmium injection does not induce metallothionein synthesis, though cadmium is avidly sequestered by pre-existing metallothionein. The differences in the induction of metallothionein by these divalent cations can be explained by the differences in their binding affinities for thiol groups in intracellular metallothionein.     

Licht- und elektronenmikroskopische Untersuchungen über die Beeinflussung der Dynamik neurosekretorischer Zellen von Enchytraeus (Oligochaeta) durch Aktinomycin D

Zusammenfassung Die Wirkung von Aktinomycin auf die neurosekretorischen Q-und P-Zellen im Cerebralganglion von Enchytraeus wurde untersucht. Die Cytophotometrie lichtmikroskopischer Präparate von Q-Zellen ergab, daß in den ersten Stunden nach Aktinomycin-Behandlung eine deutliche Verminderung PAF-positiven Materials auftritt. Die ersten Veränderungen wurden elektronenmikroskopisch zwischen 1 und 4 Std nach Aktinomycin-Injektion beobachtet. Sie waren in beiden Zelltypen am eindeutigsten am Nucleolus. Es kommt zu einer Sonderung und räumlichen Trennung von granulärem und fibrillärem Material. Letzteres wird sehr stark vermehrt.In bezug auf Veränderungen der Strukturen des Cytoplasmas unterscheiden sich die Q-und P-Zellen besonders im Verhalten des Golgi-Apparates und der Ribosomen. Der Golgi-Apparat wird in den Q-Zellen kurze Zeit nach Applikation von Aktinomycin reduziert. In den P-Zellen persistiert er dagegen über alle beobachteten Zeitstufen hinweg. Die Ribosomen lösen sich von den Membranen in den Q-Zellen 4–8 Std nach Injektion, was in den P-Zellen nicht festzustellen ist. Diese Tatsachen führen zu der Annahme, daß das System der Proteinsynthese der P-Zellen relativ stabiler als das der Q-Zellen ist.Die in den späteren Zeitstufen beobachtete Normalisierung der Zellstrukturen läßt darauf schließen, daß die Wirkung des einmalig injizierten Aktinomycins 24 Std danach nachzulassen beginnt.

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Light and electron microscopic studies on the influence of actinomycin D on the dynamics of neurosecretory cells of Enchytraeus (Oligochaeta)

Summary The influence of actinomycin on the neurosecretory Q and P cells of the brain of Enchytraeus was studied. Cytophotometrical measurements of Q cells in light mirocscopic preparations showed a significant decrease of PAF-positive material in the first hours after actinomycin application. At the ultrastructural level primary changes were established one to four hours after injection of actinomycin: In the nucleolus granular and fibrillar material became separated; there was a substantial increase of the fibrillar component.Concerning structural changes of the cytoplasm, Q and P cells differed especially with respect to the Golgi apparatus and the ribosomes. In the Q cells the Golgi apparatus had become greatly reduced shortly after actinomycin treatment. However, it persisted in P cells during all stages examined. Ribosomes became detached from membranes only in Q cells between 4 and 8 hours after injection.These data indicate that protein synthesis in P cells shows greater stability than in Q cells. The restitution of normal ultrastruoture during subsequent stages indicates that effects begin to subside 24 hours after a single injection.

Für technische Unterstützung danken wir Frl. B. Reymann, Frl. A. Zinßer, Frau B. Cosack und Frau E. Wolschner.