Diurnal changes in actin mRNA levels and incorporation of35S-methionine into actin in the rat hypothalamus

1. The in vitro incorporation of 35S-methionine into actin and total soluble proteins, as well as the levels of actin mRNA, were studied in the hypothalamus and frontal cerebral cortex of adult male rats killed at six different time intervals during a 24-hr cycle. 2. The specific activity of total soluble proteins after labeled methionine incubations did not vary as a function of time of day in any of the examined brain regions. 3. The incorporation of 35S-methionine into a 43-kDa protein, corresponding to the electrophoretic mobility of actin, varied diurnally in the hypothalamus, exhibiting a maximum at 1200 hr. Such a diurnal variation was not found in frontal cerebral cortex. 4. Similar results were obtained when labeled methionine incorporation into actin was assessed in hypothalamus and cerebral cortex by an immunoprecipitation procedure. 5. An increase in actin hypothalamic mRNA levels, quantitated by dot-blot analysis, was found at 0800, 4 hr in advance to the maximum in 35S-methionine incorporation to actin. 6. The levels of actin mRNA did not vary significantly as a function of time of day in the frontal cerebral cortex.     

An analysis of the influence of thyroid hormone on the synthesis of proteins in the tail fin of bullfrog tadpoles

By incubation of explants of tail fin from tadpoles of Rana catesbeiana in a solution of 35S-methionine for 4 h, newly synthesized proteins were labeled isotopically. After separation by two-dimensional polyacrylamide gel electrophoresis, those proteins were visualized by fluorography. Exposure of explants to culture medium containing thyroxine (T4) (150 nM) increased the incorporation of 35S-methionine into several proteins with 48 h. Effects of T4 on the relative abundance of two of these newly synthesized proteins were detected after 8 h of hormonal treatment. Very similar patterns of newly synthesized proteins were observed when proteins from explants of tail fin removed from tadpoles at metamorphic climax and immediately incubated with 35S-methionine were compared with proteins produced in fin derived from premetamorphic animals. These results are interpreted to indicate that both treatment of explants with T4 and elevation of endogenous levels of thyroid hormones during spontaneous metamorphosis increased the relative rates of synthesis of several identical proteins. The potential involvement of those proteins in early phases of metamorphic action which eventually lead to cell death and resorption is discussed.     

Change in the initiation of DNA synthesis on the nuclear matrix and its DNA-protein composition in gamma-irradiated hepatoma cells

It was shown that gamma-irradiation of Zajdela hepatoma cells (10 Gy) induces inhibition of DNA synthesis initiation at a nuclear matrix and a change in its DNA-protein content. Irradiation of hepatoma cells with 10 and 50 Gy decreases incorporation of newly synthesized proteins in the firmly bound DNA-protein complexes of nuclear matrix. After 60-120 min postirradiation incubation of cells at 37 degrees C DNA-protein content of the nuclear matrix and its firmly bound DNA-protein complexes are restored. However the rate of DNA synthesis initiation being below the control level.     

Granulocyte macrophage-colony stimulating factor stimulates the synthesis of membrane and nuclear proteins in murine neutrophils

The effect of granulocyte macrophage-colony stimulating factor (GM-CSF), a well-characterized hemopoietic regulator, on protein synthesis in murine bone marrow neutrophils is described. Bone marrow neutrophils in excess of 95% purity were obtained by fluorescence-activated cell sorting. While GM-CSF did not appear to slow the rate of dying of peritoneal exudate neutorphils or thymus cells, the viability of bone marrow neutrophils after 17 hr was enhanced (40%) by GM-CSF. GM-CFS had no effect on total 35S-methionine incorporation by thymocytes or peritoneal exudate neutrophils over a 17-hr incubation period; however, bone marrow neutrophils showed increased incorporation (approximately 10%) at all times between 5-17 hr. As viability and 35S-methionine incorporation of bone marrow neutrophils at 5 hr were minimally affected by GM-CSF, this time point was chosen to study the effect of GM-CSF on the synthesis of particular proteins. Two-dimensional polyacrylamide gels of 35S-methionine-labelled lysates were prepared from whole cells, isolated nuclei, and membranes. Quantitative analysis of the fluorograms obtained from the two-dimensional electropherograms by a computer-linked optical data digitiser indicated that out of a total of 180 proteins, the amount of label contained in 11 proteins was significantly higher in the presence of GM-CSF, while three proteins, apparently of cytoplasmic origin, contained less label than control cells. Eight of these proteins were identified as nuclear, and one was membrane derived. Attempts have been made to identify some of the inducible proteins and to correlate results with other studies of normal hemopoietic and leukemic cells. The significance and multiple functions of GM-CSF in hemopoiesis are discussed.     

Analysis of protein synthesis in morphologically classified bovine follicular oocytes before and after maturation in vitro

Bovine cumulus oocyte complexes (COCs) were isolated from antral ovarian follicles (4-8 mm). Immature COCs were classified into four categories, based on the homogeneity and clearness of the ooplasm and the transparency and compactness of the cumulus investment. In this study, the incorporation of TCA-precipitable 35S-methionine and the protein synthesis patterns of oocytes of these four categories were examined. Before maturation in vitro, similar incorporation rates and identical protein synthesis patterns were observed between oocytes of categories 1-3. Immature oocytes of category 4 showed reduced incorporation rates and exhibited aberrant protein synthesis patterns. After maturation in vitro, the patterns of category 4 oocytes were identical with the patterns of those in categories 1-3. The incorporation of 35S-methionine into in vitro matured oocytes was lower (P less than .001) in all categories. Based on these results, it is concluded that the initial classification of oocytes into four categories can be reduced to two categories.     

In Vitro Protein-Synthesizing Activity of Vesicular Stomatitis Virus-Infected Cell Extracts

Crude cytoplasmic extracts from vesicular stomatitis virus (VSV)-infected HeLa cells incorporate radioactive amino acids into hot trichloroacetic acid-precipitable material linearly for 10 to 20 min. The material synthesized in vitro corresponds in molecular weight to four of the five VSV structural proteins. However, synthesis of the viral glycoprotein (G) is significantly reduced, whereas the relative amounts of viral structural proteins L and NS synthesized are increased compared with the ratio of the proteins found in the virion. Fractionation of a VSV-infected crude cytoplasmic extract into a cytoplasmic pellet (20,000 x g for 30 min) and a cytoplasmic supernatant results in a significant reduction in protein synthesizing activity of both fractions, although both contain polysomes. The products synthesized by a cytoplasmic supernatant-directed system included all the VSV structural proteins except the glycoprotein, whereas in an in vitro system directed by the cytoplasmic pellet there is a marked reduction in synthesis of the nucleoprotein (N) and also a small relative increase in synthesis of the glycoprotein. Addition of uninfected, preincubated HeLa or L-cell S10 or a HeLa ribosomal fraction to the VSV-infected cytoplasmic pellet results in a 30- to 60-fold stimulation of (35)S-methionine incorporation. However, these uninfected extracts do not stimulate (35)S-methionine incorporation by the infected crude cytoplasmic extract or the cytoplasmic supernatant. The products synthesized by the stimulated cytoplasmic pellet now include sizeable amounts of the glycoprotein in addition to the other VSV structural proteins.     

Modification of nuclear matrix proteins by ADP-ribosylation. Association of nuclear ADP-ribosyltransferase with the nuclear matrix

Nuclear matrices were isolated by treatment of isolated HeLa cell nuclei with high DNase I, pancreatic RNase and salt concentrations. ADP-ribosylated nuclear matrix proteins were identified by electrophoresis, blotting and autoradiography. In one experimental approach nuclear matrix proteins were labeled by exposure of permeabilized cells to the labeled precursor [32P]NAD. Alternatively, the cellular proteins were prelabeled with [35S]methionine and the ADP-ribosylated nuclear matrix proteins separated by aminophenyl boronate column chromatography. By both methods bands of modified proteins, though with differing intensities, were detected at 41, 43, 46, 51, 60, 64, 69, 73, 116, 140, 220 and 300 kDa. Approximately 2% of the total nuclear ADP-ribosyltransferase activity, but only 0.07% of the nuclear DNA, was tightly associated with the isolated nuclear matrix. The matrix-associated enzyme catalyzes the incorporation of [32P]ADP-ribose into acid-insoluble products of molecular mass 116 kDa and above, in a 3-aminobenzamide-inhibited, time-dependent reaction. The possible function of ADP-ribosylation of nuclear matrix proteins and of the attachment of ADP-ribosyltransferase to the nuclear matrix in the regulation of matrix-associated biochemical processes is discussed.     

Measurement of in vivo incorporation of L-methionine-35S into cerebral proteins by quantitative autoradiography in freely-moving or anesthetized rats

Incorporation of L-35S-methionine in brain proteins was measured by quantitative autoradiography in the rat. This model was checked by biochemical procedures. In awake freely moving rat, methionine incorporation rates vary from 36 to 258 nmol/100g/min. During anaesthesia, protein synthesis in brain decreases (30 to 50%).     

Synthesis and secretion of stage-specific proteins by peri-implantation mouse embryos

Blastocysts were recovered from intact mice at various times on the fourth and fifth days of pregnancy and incubated in vitro with 35S-methionine. Labeled proteins synthesized by the embryos and secreted into the medium were separated in two-dimensions on polyacrylamide gels by electrophoresis and localized by fluorography. The array of proteins synthesized and secreted by late stage blastocysts was found to be qualitatively and quantitatively different from those released by embryos at earlier stages of development. Similar changes were also observed in secreted proteins when delayed-implanting embryos were reactivated after an injection of estrogen. Furthermore, there was a temporal correlation between the appearance of certain proteins secreted by the embryos and changes in specific proteins synthesized and released by the uterus. It is suggested that these various secreted proteins constitute a signal-response mechanism that is important for the process of embryo implantation in mice.     

Incorporation of methionine-35S into the structure of the brain at different pituitary-adrenal system hormone levels

The rate of 35S-methionine incorporation into the anterior and posterior hypothalamus, tonsil and hippocamp was studied in experiments on intact, adrenalectomized, and hypophysectomized rats as well as in those exposed to ACTH or dexamethasone. A conclusion is drawn that ACTH and glucocorticoids exert a dissimilar and non-equivalent effect on the brain structures responsible for the activity displayed by the hypophyseal-adrenal system.     

Incorporation of radiolabelled amino acids by adult Schistosoma japonicum: further characterization of a putative eggshell precursor protein.

The synthesis patterns of female-specific proteins of Schistosoma japonicum were further investigated with particular reference to the 34 kDa putative eggshell precursor protein. Adult male and female worms of S. japonicum were metabolically labelled with 14C-tyrosine, 14C-glycine and 35S-methionine in vitro. The rates of amino acid incorporation for female worms were significantly higher than for males in all radiolabelling experiments. Labelled proteins were resolved by two-dimensional gel electrophoresis and visualized by fluorography. By using 14C-tyrosine and 14C-glycine, the 34 kDa female protein band resolved into three major spots with pI 6.0, 5.8 and 5.6. On the other hand, labelling studies using 35S-methionine failed to reveal synthesis of any corresponding spots at Mr 34 kDa. These results, together with the observations that eggshell hydrolysates are very rich in glycine but poor in methionine, suggested that the 34 kDa putative eggshell precursor protein of S. japonicum consists of at least three isoelectric forms. In addition, we have demonstrated several other female-specific polypeptides synthesized by this worm.     

35S-Methionine Incorporates Differentially into Polypeptides across Leaves of Spinach (Spinacia oleracea)

The distribution of proteins across leaves may have significantimpact on optimal photosynthetic performance of leaves, howeverlittle is known about the distribution of proteins and proteinsynthesis across C₃ leaves. We report here a detailed investigationof ³⁵S-methionine incorporation into polypeptides and the steady-statepolypeptide profiles at different leaf depths across spinachleaves. About 10 highly incorporating polypeptides (three with apparentmolecular masses of 23 kDa, 21 kDa and 17 kDa were especiallydominant) were detected in a few medial leaf sections. Thesehighly incorporating polypeptides were soluble proteins, exceptfor the 17 kDa polypeptide, which was associated with thylakoidmembranes. All of the highly incorporating polypeptides werenuclearly encoded. Light significantly enhanced ³⁵S-methionineincorporation into the highly incorporating polypeptides in"sun" grown leaves, but not in "shade" grown leaves. Microautoradiographyshowed that the highly incorporating polypeptides were associatedmainly with the phloem tissue. A specific identity or functionfor the polypeptides is not known. The concentration of most polypeptides on an areal basis appearedto increase with leaf depth from the adaxial leaf surface, reachinga maximum around 25% of the leaf depth, and then declined graduallytowards the abaxial surface. The periphery of cells exhibitedhigh levels of ³⁵S-methionine incorporation, and microautoradiographyshowed that the label was mainly located in the symplast. Ingeneral, polypeptides exhibited higher rates of ³⁵S-methionineincorporation in the palisade mesophyll than in the spongy mesophy,probably due to cytoplasmic density and light. The data showthat it may be possible to study vascular bundle proteins usingparadermal leaf sections. In addition, we now can investigatehow factors such as light or CO₂ might control protein distributionacross leaves, and further explore the complex interactionsamong photosynthesis, leaf anatomy, and light. ¹The research was supported by grants from the Competitive ResearchGrants Office, U.S. Department of Agriculture (No. 91-37100-6672and No. 93-37100-8855).     

Gonadotropin releasing hormone (GnRH) stimulates both secretion and synthesis of human chorionic gonadotropin (hCG) by first trimester human placental minces in vitro

An in vitro system using the minces of placental villi from first trimester human pregnancy (6-10 weeks) has been validated to examine the effect of addition of GnRH and its analogues on hCG secreted into the medium. Addition of low concentration of GnRH or its analogues (1 X 10(-8) M to 1 X 10(-6) M) resulted in an increase in the quantity of hCG in the medium, while addition of high concentrations of GnRH resulted in an inhibitory effect. Of the analogues tested, Buserelin was highly effective in exerting an inhibitory effect. A significant increase in 35S-methionine incorporation into immunoprecipitable hCG was noticed in the presence of GnRH. These results suggests that GnRH stimulates both synthesis and secretion of hCG by first trimester human placenta.     

Incorporation of methylsulfonylmethane sulfur into guinea pig serum proteins

Methionine, an essential amino acid, and cysteine are the major sulfur-containing amino acids in the body and both are thought to be synthesized predominantly in plants and micro-organisms. Methylsulfonylmethane (MSM) is a natural constituent of the environment in which it is found in plants, in milk and urine of both bovines and humans, is a normal oxidation product of dimethyl sulfoxide (DMSO) also in the natural environment and may be part of the natural global sulfur cycle. To determine whether sulfur from methylsulfonylmethane (MSM) is incorporated into sulfur amino acids, I fed 35S-MSM to guinea pigs. 35S was incorporated into peptidyl methionine and cysteine of guinea pig serum proteins. The specific activity of 35S-methionine was 30% greater than for 35S-cysteine, suggesting a precursor-product relationship. Total specific activity of serum proteins was increased by only 30% with a 100% increase of administered 35S-MSM, suggesting a limiting step in synthesis. Approximately 1% of the radioactivity was recovered in serum proteins, none in the feces and most was excreted in the urine. Microorganisms of intestinal lumen may be responsible for the incorporation of the 35S of MSM into sulfur amino acids. MSM may provide a source of sulfur for essential animal methionine by mechanisms not yet elucidated in either animals or micro-organisms.     

Synthesis and phosphorylation of histone H1 and high mobility group proteins in the regenerating rat liver after X irradiation

A rat was irradiated to the upper abdomen including the liver and then partially hepatectomized. The subsequent synthesis and phosphorylation of histone H1 and nonhistone chromosomal high mobility group (HMG) proteins were investigated. Incorporation of [3H]lysine into histone H1 was increased and reached its peak at 27 h after hepatectomy, and 14 Gy of X rays inhibited the increase. Increase in the incorporation of [3H]lysine into HMG (1 + 2), 14, and 17 which occurred around 27 h after hepatectomy was not inhibited by 14 Gy irradiation. Phosphorylation of histone H1, measured with 32Pi incorporation in vivo, was maximal between 21 and 24 h, and it was inhibited by 4.8 Gy of X rays and delayed with 1.9 Gy. Phosphorylation of HMG 14, which was the only HMG protein phosphorylated under present conditions, was not affected by X irradiation. The [3H]thymidine incorporation into nuclear DNA started increasing at 21 h and reached its maximum at 27 h after hepatectomy. X irradiation with 4.8 Gy inhibited the incorporation, and 1.9 Gy lowered it.     

Lymphocyte migration in internally irradiated animals. Effect of internal beta radiation on the migration of spleen lymphocytes in CBA mice

The syngeneic transfer system was used to study migration of 51Cr-labelled spleen lymphocytes in mice after incorporation of beta-emitter, 35S-methionine. Migration of 51Cr-labelled lymphocytes to lymph nodes was stably decreased, and to liver, kidneys and lungs increased. The lymphocyte migration impairment was associated with the influence of beta-radiation on both the migratory properties of cells and the factors of their microenvironment responsible for the lymphocyte migration within the mouse body. No distinctions were observed in the character and manifestation of disturbances of the lymphocyte migration after the injection of 35S-methionine and gamma-emitter, 75Se-selenomethionine.     

Phosphorylation of prostatic nuclear matrix proteins is under androgenic control

Nuclear matrix fraction was isolated from rat ventral prostatic nuclei previously incubated with [gamma-32P]ATP to label nuclear phosphoproteins with 32P. A significant portion of the radioactivity was recovered in the phosphoproteins intrinsic to the nuclear matrix fraction. At 12 h after androgen deprivation (i.e., when a significant portion of the nuclear androgen receptor was known to be depleted), the rate, but not the extent, of phosphorylation of nuclear proteins (predominantly nonhistone proteins) was markedly reduced. Nuclear matrix fraction isolated from such preparations demonstrated a profound reduction in the rate of incorporation of 32P into the matrix-associated proteins without any apparent change in the gel electrophoretic profile of these proteins. The results indicate that the cAMP-independent protein kinase activity which catalyzes the phosphorylation of nuclear matrix proteins is under androgenic control. This may be germane to nuclear matrix-associated initial events in androgen action.     

Rapid and selective effects of thyrotropin on [35S]methionine incorporation into cytoplasmic and nuclear proteins in calf thyroid tissue slices

Previous studies have indicated that thyrotropin may induce general increases in RNA and protein synthesis in calf thyroid tissue slices. In this report, we show that thyrotropin selectively stimulates [35S]methionine incorporation into small numbers of specific cytoplasmic and nuclear proteins. We provide data on the time course of stimulation and on relative molecular masses and isoelectric points of hormone-response proteins. Calf thyroid tissue slices were incubated for 3 h, 6 h or 9 h in the presence or absence of thyrotropin (50 mU/ml); [35S]methionine (50-75 microCi/ml) was added for the final 3 h of incubation. Cytoplasmic and nuclear fractions were then prepared, and analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography. Thyrotropin increased [35S]methionine incorporation into two cytoplasmic and four nuclear proteins within 3 h; hormonal effects on the labeling of five of these six proteins were transient, and no longer evident after 6-9 h. In contrast, a second group of two cytoplasmic and four nuclear proteins exhibited increased labeling after a delay of 6-9 h. Our results suggest that thyrotropin selectively stimulates the synthesis of specific cytoplasmic and nuclear proteins in calf thyroid tissue slices, and that stimulation involves at least two mechanisms (one rapid, the other delayed).     

Synthesis of stress proteins under normal and heat shock conditions in gill tissue of sea mussels (Mytilus edulis) after chronic exposure to cadmium.

1. Sea mussels were exposed to 16.5 micrograms Cd/1 under semi-field conditions for almost one year. The isolated gills were incubated with 35S-methionine or -cysteine. 2. Chronic exposure to cadmium neither altered the rate of amino acid incorporation nor induced expression of heat shock proteins in the gills. 3. Heat shock imposed after chronic exposure to cadmium resulted in an increased synthesis of heat shock proteins, especially those of high molecular weight. 4. Synthesis of cadmium-binding, low molecular weight proteins was observed at any point of the exposure time. Their cadmium-binding capacity and rate of synthesis, after the initial increase, remained unchanged throughout the exposure.