Sequence of molecular events involved in induction of allophanate hydrolase

Addition of urea to an uninduced culture of Saccharomyces at 22 C results in appearance of allophanate hydrolase activity after a lag of 12 min. We have previously demonstrated that both ribonucleic acid (RNA) and protein synthesis are needed for this induction to occur. To elucidate the time intervals occupied by known processes involved in induction, temperature-sensitive mutants defective in messenger RNA transport from nucleus to cytoplasm (rna1) and in protein synthesis initiation (prt1) were employed along with an RNA polymerase inhibitor in experiments that measure cumulative synthetic capacity to produce allophanate hydrolase. These measurements identify the time within the lag period at which each of the above processes is completed. We observed that RNA synthesis, rna1 gene product function, and protein synthesis initiation are completed at 1 to 1.5, 4, and 9 to 10 min, respectively.     

Effect of hydrocortisone and actinomycin D at different time of the day on incorporation of labeled precursor into the nuclear proteins of intact rat liver

Variations in the incorporation of labelled precursor into nuclear rat liver proteins during a day in normal conditions and in the presence of hydrocortisone and actinomycin D are studied. Synthesis of nuclear proteins is found to have a fluctuating character. Hydrocortisone and actinomycin D, injected at different day time, induce changes in the character of nuclear proteins synthesis , which are more pronounced for histones. The effect of the hormone and the antibiotic is tightly bound with the functional state of liver cells at given moment.     

An assay for albumin messenger RNA in an vitro protein synthesizing system from wheat germ

The synthesis of serum albumin in an in vitro protein-synthesizing system from wheat germ stimulated with rat liver polysomal RNA is demonstrated by immunoprecipitation. The newly synthesized albumin has the same electrophoretic mobility as rat serum albumin. There is a linear increase in precursor incorporation into total protein and albumin with increasing RNA concentration. Potassium and magnesium optima for albumin synthesis are different from those for total protein synthesis.     

Lead suppresses chimeric human transferrin gene expression in transgenic mouse liver

The major iron-transport protein in serum is transferrin (TF) which also has the capacity to transport other metals. This report presents evidence that synthesis of human TF can be regulated by the metal lead. Transgenic mice carrying chimeric human TF-chloramphenicol acetyl transferase (CAT) genes received lead or sodium salts by intraperitoneal injections or in drinking water. Transgene expression in liver was suppressed 31 to 50% by the lead treatment. Lead regulates human TF transgenes at the mRNA level since liver CAT enzyme activity, CAT protein, and TF-CAT mRNA levels were all suppressed. The dosages of lead did not alter synthesis of the other liver proteins, mouse TF and albumin, as measured by Northern blot analysis of total liver RNA and rocket immunoelectrophoresis of mouse sera. Moderate levels of lead exposure were sufficient to evoke the human TF transgene response; blood lead levels in mice that received lead acetate in drinking water ranged from 30 micrograms/dl to 56 micrograms/dl. In addition to suppressing expression of TF-CAT genes in transgenic mice, lead also suppressed synthesis of TF protein in cultured human hepatoma HepG2 cells. The regulation of human TF apparently differs from the regulation of mouse TF which is unresponsive to lead exposure.     

From laboratory studies to clinical trials: past benefits and future directions in hyperthermia

Three examples of clinical research trials that were direct outgrowths of hyperthermia laboratory investigations performed at Stanford University, as well as elsewhere, are reviewed and directions for future studies presented. The first series of laboratory-clinical studies investigated the influence of the number of hyperthermia treatments on tumour response in combined radiotherapy-hyperthermia regimens. A prospective randomized clinical trial was performed and showed no difference in response rate or duration of local control in combined radiation therapy-hyperthermia regimens comparing two versus six hyperthermia treatments. The results were in agreement with the laboratory studies in RIF murine tumours which suggested that persistent thermotolerance limited the effectiveness of multiple heat treatments in fractionated hyperthermia treatment regimens. The second prospective clinical trial was undertaken to investigate the influence of pretreatment tumour temperatures on subsequent response to radiotherapy and hyperthermia. In agreement with the laboratory studies, low (< 37 degrees C) pretreatment temperatures sensitized superficially located tumours to subsequent hyperthermia treatment. Lower pretreatment temperatures and larger differentials between minimum treatment temperatures and pretreatment maximum or mean temperatures were correlated with the duration of local control. The final studies investigated thermosensitizing agents, agents which were non-toxic at 37 degrees C but became cytotoxic at elevated temperatures. Thermosensitizers such as sulphydryl compounds have demonstrated up to 10,000-fold enhancement of cell killing at exposure to 43 degrees C for 1 h and may be considered for tumour sensitization. However, normal tissue may also be sensitized as was noted for topical anaesthetics such as lidocaine.(ABSTRACT TRUNCATED AT 250 WORDS)     

The quantity of dietary protein affects brain protein synthesis rate in aged rats

The purpose of this study was to determine whether the quantity of dietary protein affects the rate of brain protein synthesis in aged rats. Experiments were conducted on three groups of 30-wk-old rats fed diets containing 0, 5 or 20 g casein/100 g for 10 d. The fractional rates of protein synthesis in brain, liver and kidney declined with a decrease in quantity of dietary protein. In brain, liver and kidney, RNA activity [g protein synthesized/(g RNA.d)] was significantly correlated with the fractional rate of protein synthesis. The RNA concentration (mg RNA/g protein) was not related to the fractional rate of protein synthesis in any organ. The results suggest that the rate of protein synthesis in the brain declines with a decrease in quantity of dietary protein in aged rats, and that RNA activity is at least partly related to the fractional rate of brain protein synthesis.     

Studies on transmembrane and paracellular phenomena in the foot of the slug agriolimax reticulatus (Mü)

Employing livers from rats fed on a protein-free diet for two weeks, the effects of protein deficiency on both biosynthesis and degradation of rRNA were investigated and the following results were obtained. 1. Protein deficiency led to a decrease of total liver RNA content per DNA to about 80% of that in normal rat liver. 2. From the kinetics of rRNA labelling with [14C]orotic acid in vivo, the half-lives of cytoplasmic rRNA's of normal and protein-deficient rat livers were determined to be 6.2 and 5.1 days, respectively. Furthermore, considering the pool size of rRNA in rat liver, the turnover rate of cytoplasmic rRNA was calculated to be 0.212 pmole/min/mg of nuclear DNA in normal rats and 0.240 pmole/min/mg of nuclear DNA in protein-deficient rats. 3. From the electrophoretic patterns of nucleolar RNA's of both groups of rat livers labeled with [14C]orotic acid, the time courses of the specific activities of nucleolar 45S, 32S, and 28S rRNA's were analysed and the half-life of each nucleolar RNA in both groups of rat livers was determined. Nucleolar 45S, 32S, and 28S RNA's had half-lives of 6.0, 15.9, and 26.5 min in normal rats, respectively, and 5.5, 19.4, and 22.9 min in protein-deficient rats, respectively Considering the pool size of each nucleolar RNA obtained from the leectrophoretic pattern, the turnover rates of 45S, 32S, and 28S RNA's were calculated to be the same, i.e., o.189 pmoles/min/mg of nuclear DNA, in normal rat liver and 0.372, 0.372, and 0.358 pmoles/min/mg of nuclear DNA in protein-deficient rat liver, respectively. 4. These results indicate that protein deficiency increased both the rate of degradation of cytoplasmic rRNA and that of nucleolar rRNA synthesis in rat liver. While in normal rat liver the rates of rRNA synthesis and degradation were rather similar, the rate of rRNA synthesis in protein-deficient rats was about 1.5 times higher than that of its degradation. Therefore, the decrease of total liver RNA content in protein deficiency might be accounted for by stimulated degradation of rRNA in the nucleus. 5. The activities of RNase in nuclear fractions of both groups of rat livers were compared. Both activities of nuclear acid RNase and especially that of the free form of alkaline RNase in protein-deficient rat liver were higher than those in normal rat liver.     

Effect of a thyroid hormone treatment on brain protein synthesis in rats

The effect of the thyroid hormone on the rate of brain protein synthesis in rats was studied. Experiments were conducted on three groups of rats given 6-propyl-2-thiouracil (PTU, a thyroid inhibitor) without a triiodothyronine (T3) treatment, those treated with PTU + T3, and those treated with neither PTU nor T3 (control). The fractional rates of protein synthesis in the brain, liver, and kidney of rats given PTU + T3 were significantly greater than those in rats given PTU alone. In the brain and kidney, the RNA activity [g of protein synthesized/(g of RNA.d)] were significantly correlated with the fractional rates of protein synthesis. In the liver and kidney, the RNA concentration (mg of RNA/g of protein) was related to the fractional rate of protein synthesis. These results suggest that the thyroid hormone treatment would be likely to increase the rate of protein synthesis in the brain of rats, and that the RNA activity is, at least partly, related to the fractional rate of brain protein synthesis.     

Deprivation of dietary nucleotides decreases protein synthesis in the liver and small intestine in rats

BACKGROUND & AIMS: Dietary nucleotides are reported to influence the growth and functioning of the liver and small intestine. The aim of this study was to examine the mechanism by which nucleotides exert their effects in these tissues by assessing protein synthesis activity and related parameters in the presence or absence of dietary nucleotides. METHODS: Rats were fed a purified diet with or without nucleotides for 10 days. Fractional protein synthesis rate, RNA and DNA concentrations, polysome size distribution, and number of ribosomes were assessed. RESULTS: Fractional protein synthesis rates of the liver and small intestine were lower in the nucleotide-deprived group than in the control group. In the liver, RNA concentration was also lower in the nucleotide-deprived group, but values in the small intestine were similar in the two groups. In the liver, deprivation of nucleotides resulted in a reduction in the number of ribosomes and in polysome breakdown. Protein and DNA concentrations did not vary in the liver; however, the concentration of DNA was lower in the small intestine of the nucleotide-deprived group than in the control group. CONCLUSIONS: Dietary nucleotides can modulate protein synthesis in the liver and small intestine as a result of tissue-specific nucleic acid changes.     

The effect of hyperthermia alone or in combination with actinomycin D on the RNA metabolism of solid tumors in children

The incorporation of 3H-uridine into the RNA was studied under normothermia 37 degrees C/120 min, hyperthermia 42.5 degrees/120 min, and both in combination with Actinomycin D by an autoradiographic in vitro method in 19 solid tumors of children: 6 Wilms' tumors, 5 neuroblastomas, 4 osteogenic sarcomas, and 4 different tumors. Hyperthermia invariably reduces the 3H-uridine incorporation into RNA by 11.7--86.4%, with an average of 47.5%. Actinomycin D consistently inhibits the 3H-uridine incorporation between 27.7 and 99.8%, with the average inhibition of 62.0% being far greater than that recorded for hyperthermia. The highest degree of 3H-uridine incorporation inhibition is obtained using hyperthermia in combination with Actinomycin D. The inhibition varies from 45.5--99.8%, with an average of 81.4%. In spite of the general regularity, the effect of hyperthermia and Actinomycin D are characterized by individual patterns. Obviously, they are dependent on proliferative activity rather than upon the particular type of tumor. The use of supranormal temperatures for the treatment of malignant tumors in man, also in combination with radiation or cytostatic drugs, is a possible and promising method of therapy.     

RNA synthesis in alpha-amanitin-poisoned rats: prevention of recovery by inhibition of protein synthesis

The treatment with cycloheximide of rats previously poisoned with alpha-amanitin hinders the recovery of RNA synthesis observed in the liver of rats treated with alpha-amanitin alone. The recovery of RNA synthesis can be ascribed to the capability of poisoned rats to synthesize new RNA-polymerase II.     

Nuclear RNA in the mouse spleen at the peak of primary immune response

Results of studies on the synthesis of different fractions of cytoplasmic RNA in the spleens of immunized mice at the peak of primary immune response are reported. RNA was extracted by the phenol-detergent procedure from the cytoplasm of spleen cells of mice immunized with sheep red blood cells. The kinetics of labeling in vivo with 32P-orthophosphate indicate an accelerated biosynthesis and transport from nucleus to cytoplasm of 28S, 18S and 4S fractions of RNA.     

RNA synthesis in spermatocytes and spermatids and preservation of meiotic RNA during spermiogenesis in the mouse

The rate of RNA synthesis at different stages of spermatogenesis in the mouse, and the preservation of RNA from the diploid to the haploid phase of spermatogenesis were studied in homogeneous germ cell fractions separated by velocity sedimentation at unit gravity. The uridine pool expansion method was used for determining the rate of RNA synthesis: seminiferous tubules were labelled in culture with increasing concentrations of [3H]-uridine and the incorporated radioactivity was estimated in cell fractions separated by velocity sedimentation. The results indicate that before nuclear elongation, round spermatids (steps 1 to 8 of spermiogenesis) synthesize RNA at the same rate per DNA content as middle-late pachytene spermatocytes. The preservation of RNA molecules synthesized in meiosis was investigated by labelling pachytene spermatocytes with T3H]uridine in vivo and collecting samples of germ cells at definite stages of spermatogenesis at various time intervals thereafter. The results show that a considerable proportion the RNA synthesized during the pachytene stage is preserved through spermatid development until late spermiogenesis.     

Epinephrine or peplomycin combined with hyperthermia in irradiated Lewis lung carcinoma: effects on tumor growth, skin reaction, and lung metastasis

Although hyperthermia potentiates the effect of radiation, the combined effect decreases as the time between irradiation and hyperthermia increases. The purpose of this study was to prevent the rapid loss of efficacy by the local injection of epinephrine or peplomycin(PEP), two agents known as hyperthermic potentiators. In this study, Lewis lung carcinoma implanted in the foot of BDF1 mice was used for the assessment of tumor growth, skin reactions, and lung metastasis. The tumors were irradiated, then warmed in a water bath for 45 min. The retarding effects of hyperthermia on tumor growth and skin reactions were lost 2 days after irradiation. However, when PEP or epinephrine was injected before hyperthermia, tumor growth was distinctly delayed. The effect of epinephrine was greater than PEP and still showed enhancement 8 days after irradiation. For skin reactions, no significant enhancing effect was observed. Lung metastasis was significantly inhibited by the addition of epinephrine either 0 or 2 days after irradiation. In conclusion, the local administration of epinephrine combined with hyperthermia significantly retarded tumor growth without an increase in skin reactions or lung metastases. Possible mechanism underlying this phenomenon was discussed.     

Complex behavior of self-propagating reaction waves in heterogeneous media

Self-propagating high temperature reaction waves, leading to the synthesis of advanced materials, are investigated in a variety of heterogeneous reaction systems by using a digital high-speed microscopic video recording technique. It is shown that, although on the macroscopic length and time scales, the reaction appears to move in a steady mode, on the microscopic level it has a complex character that is related to the reaction mechanism.     

The effects of local hyperthermia on the catabolism of a radioiodinated chimeric monoclonal antibody

Local hyperthermia has been shown to increase the tumor uptake and tumor:normal tissue ratios of radiolabeled monoclonal antibodies (mAbs) in athymic mouse xenograft models. The current study was undertaken to determine whether this behavior was related in part to alterations in mAb catabolism by local hyperthermia. Human/mouse chimeric 81C6 mAb reactive with tenascin and a nonspecific control mAb were labeled with 125I using Iodo-Gen and given to separate groups of athymic mice bearing s.c. D-54 MG human glioma xenografts. Half of the animals were then subjected to 4-h tumor-localized hyperthermia at 41.8 degrees C, a protocol previously shown to enhance the specific tumor uptake of the mAb in this xenograft model. The tumor, serum, liver, kidney, and urine were collected from heated as well as control animals 4 and 24 h after injection of the mAb and analyzed by SDS-PAGE and trichloroacetic acid precipitation. At 24 h, a significantly higher percentage of 81C6 was present as intact mAb in the tumors harvested from heated animals compared with those from controls. Unexpectedly, intact mAb was found in the urine of mice immediately after hyperthermia, but not in unheated control animals. We conclude that local hyperthermia decreases the catabolism of the mAb in the tumor and increases the urinary excretion of the mAb through a transient increase in glomerular permeability.