Storage Protein Synthesis during Oat (Avena sativa L.) Seed Development

Oat (Avena sativa L.) seeds harvested at 2-day intervals from anthesis to maturity were tested for their ability to incorporate [³⁵S]sulfate into protein. Incorporation of [³⁵S]sulfate into TCA-insoluble material began 2 to 4 days postanthesis (DPA), reached a peak 14 to 16 DPA, and was barely detectable by 24 DPA. Incorporation of label into globulin was parallel to total protein accumulation, and averaged about 85% of the total protein synthesis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total protein extracted from developing seeds indicated that some polypeptides coinciding with the α and β globulin subunits were present 2 to 4 DPA, but the full complement of globulin polypeptides was not present until 10 DPA. Immunoprecipitation of in vivo labeled seed extracts showed that globulin polypeptides and the 59 kilodalton precursor were present at early stages of development (4 DPA). Quantitation of dot blot analysis, using an oat globulin cDNA clone as a probe, indicated that one species of oat globulin mRNA was most abundant 15 DPA, which is during the peak time of storage protein synthesis.     

The synthesis of a variant-specific antigen by Trypanosoma brucei in vitro.

A variant-specific surface antigen from a cloned population of Trypanosoma brucei S42 has been isolated and partially characterized. [35S]L-methionine was found to be incorporated into this material by cells incubated in vitro in a chemically defined medium. Incorporation of [35S]L-methionine was inhibited by cycloheximide and puromycin at concentrations which are known to specifically inhibit protein synthesis in other systems. The rate of synthesis of the variant-specific antigen in vitro has been estimated to be about 8% of the rate in vivo. Newly synthesized [35S]L-methionine-labelled variant-specific antigen was incorporated into the surface coat.     

The effect of cycloheximide on synthesis of proteoglycans by cultured chondrocytes from the Swarm rat chondrosarcoma

Proteoglycan synthesis by cultured chondrocytes from the Swarm rat chondrosarcoma was examined after treatment with 0.1 mg/ml of cycloheximide which inhibited [3H]serine incorporation into total protein by greater than 90%. Incorporation of [35S]sulfate into proteoglycans decreased with nearly first order kinetics (t 1/2 = 96 +/- 6 min) with an accompanying increase in the size of the proteoglycan molecules, primary due to an increase in chondroitin sulfate chain sizes. After 5 h of cycloheximide treatment, when [35S]sulfate incorporation was inhibited by about 90%, addition of 1 mM beta-D-xyloside restored 76% of the incorporation into chondroitin sulfate observed in cultures treated only with xyloside. This suggests that the biochemical pathways for the affected by cycloheximide treatment. Cultures were prelabeled for 15 min with either [3H]serine or [35S]-methionine, and then cycloheximide was added to block further protein synthesis. Both precursors appeared in completed proteoglycan molecules with nearly first order kinetics with t 1/2 values of 92 +/- 8 and 101 +/- 11 min for [3H]serine and [35S]methionine, respectively, values in close agreement with the t 1/2 from the [35S]sulfate data. These results suggest that after cycloheximide treatment, the rate of [35S]sulfate incorporation into proteoglycan, after a correction for increases in chondroitin sulfate chain size, was directly proportional to the size of the intracellular pool of core protein. From the steady state rate of proteoglycan synthesis (estimated to be about 80 ng/min/10(6) cells in separate experiments) and a corrected t 1/2 value of 60 min, the amount of precursor core protein can be calculated to be about 500 ng/10(6) cells in these experiments.     

Altered patterns and synthesis of extracellular matrix macromolecules in early osteoarthritis.

The synthesis and contents of extracellular non-collagenous matrix macromolecules was studied in early and late human osteoarthritic (OA) cartilage obtained at surgery for sarcomas in the lower extremities (normal and early OA) or for total knee replacement (late stage OA). The early OA samples were those that had some fibrillation in the joint by visual examination. One group had fibrillation in the area sampled and the other group had no fibrillation. Cartilage was taken from the same topographical area on the medial femoral condyle in all the samples, labeled with [3H]leucine and [35S]sulfate for 4 h at 37 degrees C and extracted with 4 M guanidine-HCl. Analysis of the extracts showed that the total amount of proteoglycans relative to hydroxyproline content was higher in the early and late OA than in the normal cartilage. These proteoglycans showed a relatively lower [35S]sulfate incorporation into GAG chains and a higher [3H]leucine incorporation. The pattern of newly synthesized proteins was altered similarly in early and late OA. Notably, synthesis of cartilage oligomeric matrix protein (COMP), fibronectin, and cartilage intermediate layer protein (CILP) was increased, also reflected in their abundance as determined by enzyme-linked immunosorbent assay (ELISA). Collagen synthesis appeared significantly increased only in the late stage OA. The observed altered composition and pattern of biosynthesis indicate that the joint undergoes metabolic alterations early in the disease process, even before there is overt fibrillation of the tissue. The early OA samples studied appear to represent two distinct groups of early lesions in different stages of the process of cartilage deterioration as shown by their differences in relative rates of synthesis and abundance of proteins.     

Inhibition of branching morphogenesis and alteration of glycosaminoglycan biosynthesis in salivary glands treated with β-d-xyloside

The role of glycosaminoglycans (GAGs) in the branching morphogenesis of embryonic mouse salivary glands was investigated by culturing the glands in the presence of xylose derivatives which stimulate synthesis of the xyloselinked classes of GAGs. Branching morphogenesis is inhibited severely, but reversibly, by 0.5–1.0 mM π-nitrophenyl-β-d-xylopyranoside and the inhibition correlates with a stimulation of incorporation of [³H]glucosamine (1.8-fold) and [³⁵S]sulfate (almost 3-fold) into GAGs. The effect of β-xyloside on accumulation of newly synthesized GAG also occurs in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the production of free GAG chains rather than proteoglycan-associated GAGs is being stimulated. The xyloside effects apparently do not result from general cytotoxicity of the derivatives, since similar concentrations of the α-anomer do not alter salivary branching or GAG synthesis, the rudiments resume morphogenesis when returned to control medium, and the effect on GAG synthesis is stimulatory rather than inhibitory. The study suggests that GAG biosynthesis plays an important role in salivary development, and that xylosides provide useful probes for characterizing the molecular events controlling branching morphogenesis.     

Detection by means of cell fusion of macromolecular synthesis involved in the reconstruction of the nuclear envelope in mitosis

Using the cultured Chinese hamster cell line Don, G1 or S or a mixture of late-S/G2 cells were prepared by release from metaphase arrest. Metaphase (M) cells were also obtained by mitotic arrest of log-phase cultures with Colcemid and held in metaphase; such M cells remained untreated with any other compound and were termed standard M cells. When interphase (I) cells were fused at pH 8.0 and 37 degrees C with standard cells in the presence of Colcemid by means of UV-inactivated Sendai virus, binucleate interphase-metaphase (I-M) cells were obtained. In a given I-M cell there occurred within 30 min after fusion either prophasing of the I nucleus or formation of a nuclear envelope (NE) around the chromosomes. About 20% of early G1 cells, 35% of cells at the G1/S boundary, 50% of S cells, and 70% of late S/G2 cells could induce NE formation. If, before fusion, cycloheximide (CHE), an inhibitor of protein synthesis, was present during release from M arrest, the cells entered G1 but not S. About 20% of such early G1 cells, like the untreated early G1 cells, had the capacity to induce NE formation during subsequent fusion. If the cells were blocked in S with 5 mM thymidine (TdR), At least 80% of these cells could induce NE formation during subsequent fusion, but in the presence of both TdR and CHE only 35% could do so. It appeared, therefore, that protein synthesis in interphase was required for NE formation. Experiments with actinomycin D indicated that RNA synthesis was also necessary for acquisition of NE-inducing capacity. About 35% of G1 cells from confluent monolayers had the NE-inducing capacity, but prolonged exposure to CHE reduced their number to 8% . Removal of CHE restored the ability while the cells still remained in G1. This result indicated that continuing protein synthesis in the G1 cell was needed for NE formation subsequent to fusion. The fact that macromolecular synthesis must occur in the I cell before fusion if NE formation was to occur in the fused I-M cell lends further support to evidence adduced earlier that this phenomenon is a normal mitotic event. Prophasing of the I nucleus in I-M cells did not appear to be dependent on macromolecular synthesis in the I cell; earlier results from this laboratory showed, however, that protein synthesis in the prior G2 period of the M cell of the I-M pair was required for prophasing.     

Sulfated glycosaminoglycans (GAG) in the developing mouse brain : Quantitative aspects on the metabolism of total and individual sulfated GAG in vivo

Sulfation and desulfation of total glycosaminoglycans (GAG) as well as of chondroitin sulfates (A + C), dermatan sulfate, and heparan sulfate were quantified in the developing cerebrum and cerebellum of mice by labeling with [35S]sulfate combined with chases started 24 hr after [35S]sulfate injection. In both the developing cerebrum and cerebellum, the rate of biosynthesis of total sulfated GAG was highest shortly after birth (2 days), decreased sharply thereafter, and reached a plateau after 14 days. The biosynthetic activities of chondroitin sulfates and heparan sulfate decreased sharply up to 14 days and retained constant levels afterward. By contrast, the rates of biosynthesis of dermatan sulfate increased up to 14 days. The biodegradation rates of total sulfated GAG as well as of chondroitin sulfates, heparan sulfate, and dermatan sulfate were strongly correlated with the corresponding rates of biosynthesis during the first 2 postnatal weeks. Total and individual sulfated GAG showed high degradation rates resulting in half-life times of a few hours up to 1 1/2 days. Thus sulfated GAG are synthesized in excess and the actual net content seems to be co-regulated to a high degree by lysosomal degradation. In both brain parts, a proportional increase of the sulfated GAG content vs the total GAG content from 40% at birth to 90% at 28 days was observed. Since during development heparan sulfate and dermatan sulfate manifested a relative increase in their daily net synthesis besides a decrease of chondroitin sulfates, a developmental increase of the sulfate groups linked to GAG is evidenced. This molecular differentiation resulting in microenvironmental changes may be of high functional significance.     

The teratogenic mechanism of 6-aminonicotinamide on limb formation of chick embryos: abnormalities in the biosynthesis of glycosaminoglycans and proteoglycans in micromelia

After a dose of 10 micrograms of 6-aminonicotinamide (6-AN) was administered to day-4- chick embryo in ovo, micromelia was obviously observed in the hind limbs of 7-day chick embryos. We examined the teratogenic mechanism of 6-AN by using the normal or micromelial hind limbs (buds) from day 5 to day 7, with special attention to the biosynthesis of glycosaminoglycan (GAG) and proteoglycan as an index of limb chondrogenesis. The present study provides evidence for abnormalities in the levels of GAG or proteoglycan biosynthesis in the micromelial hind limbs (buds). 1) Both [35S]sulfate and [3H]glucosamine incorporation into GAG per 10 limbs or mg DNA of the micromelia were inhibited, suggesting a decrease of GAG synthesis. 2) The micromelial limbs synthesized low-sulfated chondroitin sulfate (chondroitin) as judged by the 35S/3H ratio, the proportion of unsulfated disaccharide (delta Di-0S), and the result of cellulose acetate electrophoresis, although there were no significant differences in the approximate molecular size of 35S-chondroitin sulfates synthesized between the normal and micromelial limbs. 3) PAPS-synthesizing activity in the micromelial limbs was markedly inhibited, and this may result in the production of low-sulfated proteoglycan. 4) The transition from mesenchymal- to cartilage-specific proteoglycan synthesis did not appear in the micromelial limbs as judged by the sedimentation profiles. 5) 6-AN caused marked reductions in the oxygen consumption and ATP level of the micromelial limbs, thereby causing the defect in PAPS formation. We suggest that these 6-AN-induced sequential molecular defects (the reduction of respiratory activity, ATP and PAPS level, and concomitant interference with GAG and proteoglycan biosynthesis) in the limbs (buds) during the critical period of limb morphogenesis must be major factors resulting in the cartilage growth retardation or disorder, i.e., micromelia.     

INCORPORATION OF TRITIUM-LABELED THYMIDINE AND LYSINE INTO CHROMOSOMES OF CULTURED HUMAN LEUKOCYTES

The incorporation of thymidine-H³ and lysine-H³ into human leukocyte chromosomes was studied in order to determine the temporal relationships between the syntheses of chromosomal deoxyribonucleic acid and chromosomal protein. The labeled compounds were incorporated into nuclei of interphase cells. Label from both precursors became apparent over the chromosomes of dividing cells. Incorporation of thymidine-H³ occurred during a restricted period of midinterphase (S) which was preceded by a nonsynthetic period (G₁) and followed by a nonsynthetic period (G₂). Incorporation of lysine-H³ into chromosomal protein occurred throughout interphase. Grain counts made over chromosomes of dividing cells revealed that the rate of incorporation of lysine-H³ into chromosomal protein differed during various periods of interphase. The rate of incorporation was diminished during G₁. During early S period the rate of incorporation increased, reaching a peak in late S. The high rate continued into G₂. Thymidine-H³ incorporated into DNA was distributed to mitotic chromosomes of daughter cells in a manner which has been referred to as a "semi-conservative segregation." No such semi-conservative mechanism was found to affect the distribution of lysine-H³ to the mitotic chromosomes of daughter cells. Therefore, it is concluded that synthesis of chromosomal protein and its distribution to chromosomes of daughter cells are not directly influenced by synthesis and distribution of the chromosomal DNA with which the protein is associated.     

Cell-free translation in lysates from Spodoptera frugiperda (Lepidoptera: Noctuidae) cells

1. Conditions for in vitro translation of mRNA in cell-free extracts from cultured Spodoptera frugiperda cells were defined. 2. Incorporation of [35S]methionine into acid-precipitable material increased for approximately 1 hr, and was sensitive to the protein synthesis inhibitors pactamycin and cycloheximide. 3. Micrococcal nuclease-treated lysate, primed with purified rabbit globin mRNA, synthesized a major protein with the size of full length globin, indicating that the lysate supported correct initiation and elongation of polypeptides.     

Incorporation of radioactive precursors into glycosaminoglycans by rat muscle fibroblasts exposed to a solubilized rat bone matrix fraction

Confluent cultures of rat muscle fibroblastic cells respond by increased glycosaminoglycan (GAG) synthesis when cultured in medium containing a solubilized bone matrix fraction (SBM) at a concentration of 100 micrograms/ml. The metabolism of the GAG associated with the cell pellet, the cell surface and the tissue culture medium fractions was studied, in the presence and absence of SBM, by measuring the incorporation of radioactivity from [3H]glucosamine and [35S]SO4 into the isolated GAG. Net synthesis of hyaluronic acid and of chondroitin sulfate in the medium fraction increased more rapidly in cultures containing SBM compared to controls, and the accumulation of labelled GAG in the medium of the treated cultures was approximately linear with respect to the length of incubation. The addition of SBM also resulted in increased incorporation of 3H and of 35S into the GAG of the cell surface and cell pellet fractions. In these fractions, stimulation of incorporation of radioactivity occurred in two waves: an early, relatively minor increase and a later relatively major increase. The relatively major stimulation of radioactivity into the GAG of the cell surface fraction occurred between 24 and 48 h and was independent of any apparent effect of serum.     

Stimulation by glucocorticoids of the differentiated phenotype of chondrocytes and the proliferation of rabbit costal chondrocytes in culture

Hydrocortisone stimulated glycosaminoglycan (GAG) synthesis, a characteristic of the cartilage phenotype, of rabbit costal chondrocytes in confluent quiescent culture, as judged by the incorporations of [35S]sulfate and [3H]glucosamine. Hydrocortisone also stimulated incorporation of [3H]serine into proteoglycan. The stimulation of GAG synthesis by hydrocortisone was dose-dependent and maximal at a physiological concentration of 10(-7) M. Hydrocortisone also stimulated GAG synthesis in cultures in the log-phase of growth. In this case, its maximal effect was observed at a concentration of 10(-6) M. The magnitude of the increase of GAG synthesis in response to hydrocortisone was larger in confluent culture than in log-phase cultures. Hydrocortisone stimulated DNA synthesis dose-dependently, and its effect was observable at a physiological concentration. However, no stimulation of DNA synthesis by hydrocortisone was observed in serum-free medium, in contrast to that of GAG synthesis. Hydrocortisone also increased protein synthesis and the cell number. Dexamethasone also stimulated the syntheses of both GAG and DNA. These results show that glucocorticoids stimulated both the differentiated phenotype of chondrocytes and the proliferation of rabbit costal chondrocytes in culture. Moreover, the effect of glucocorticoids was primarily on the differentiated phenotype of chondrocytes and its effect on proliferation was permissive.     

Comparative Incorporation of Proenkephalin-Derived Peptides, Chromogranin A, and Dopamine β-Hydroxylase into Chromaffin Vesicles

The incorporation of enkephalin-containing peptides (ECPs) derived from proenkephalin into chromaffin vesicles was examined in primary cultures of adrenal medullary chromaffin cells. Cells were pulse-labeled with [35S]methionine and chased for periods up to 24 h. Chromaffin vesicles in cell homogenates were then fractionated by density gradient centrifugation and the presence of [35S]Met-enkephalin sequences in gradient fractions determined. 35S-ECPs were incorporated into particles suggestive of immature vesicles within 1-2 h after radiolabeling. Vesicle maturation, measured by co-equilibration of 35S-ECPs and total ECPs in the gradients, was complete within 9-12 h and was unaffected by treatments that increase proenkephalin synthesis. Incorporation of [35S]chromogranin A into chromaffin vesicles followed a similar time course, but 35S-labeled dopamine beta-hydroxylase was much more slowly incorporated, possibly reflecting differences in incorporation of membrane and soluble components. In summary, the data demonstrate that ECPs are rapidly sequestered in immature chromaffin vesicles, a process unaltered by changing rates of proenkephalin synthesis.     

Control of isocitrate lyase synthesis in Chlorella fusca var. vacuolata. Rate of enzyme synthesis in the presence and absence of acetate measured by [35S]methionine labelling and immunoprecipitation.

The rate of increase of isocitrate lyase activity was measured in darkened Chlorella fusca var. vaculoata cultures in the presence and absence of acetate and compared with the rate of incorporation of [35S]methionine into isocitrate lyase enzyme protein under the same conditions. Isocitrate lyase enzyme protein was isolated for this purpose by specific immunoprecipitation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. After 4h in the dark, in the presence of acetate the rate of increase of isocitrate lyase activity was 75 times that in the absence of acetate. Incorporation of [35S]methionine into isocitrate lyase was 140 times greater in the presence of acetate. Incorporation of [35S]methionine into the trichloroacetic acid-insoluble fraction overall was about five times as fast in the presence of acetate. These data are not consistent with an increased turnover of isocitrate lyase enzyme molecules, sufficient to account for the low rate of increase of isocitrate lyase activity in the absence of acetate. The greater rate of enzyme synthesis in the presence of acetate must therefore be due to some effect of this metabolite on the processing or translation of isocitrate lyase mRNA.     

Identification of sites of cysteine misincorporation during in vivo synthesis of bacteriophage T7 0.3 protein

The 0.3 protein encoded by coliphage T7 does not normally contain cysteine residues. Incorporation of [35S]cysteine can therefore be used to assay mistranslation. We have purified 0.3 protein, synthesized in the presence of [35S]cysteine, from T7 infected cells of E. coli and determined the locations of misincorporated cysteine residues. Analysis of the molecular weights (Mr) of [35S]cysteine-labeled tryptic peptides of 0.3 protein demonstrated that cysteine (encoded by UGU or UGC) is not extensively misincorporated, as might be predicted by substitution for arginine residues (encoded by CGU or CGC). Edman degradation of the amino-terminal 50 residues of [35S]cysteine-labeled 0.3 protein determined that cysteine was most frequently misincorporated at position 15, which is correctly occupied by a tyrosine residue (encoded by UAC). There are four other tyrosine codons (1 UAU; 3 UAC) in the region of the 0.3 protein studied, but these were not mistranslated. The context in which a codon is located must therefore be more important in causing mistranslation than the sequence of the codon itself. Misincorporation of [35S]cysteine was also found at positions 9 (ACC, asparagine), 16 (GAA, glutamic acid), 41 (GCC, alanine) and 42 (GAU, aspartic acid). One mistranslation event appears to increase the likelihood that the following codon will also be mistranslated. This clustering of misincorporated [35S]cysteine residues was accentuated in 0.3 protein synthesized in the presence of streptomycin.     

Expression of the mitochondrial genome in HeLa cells. XXI. Mitochondrial protein synthesis during the cell cycle

Chloramphenicol sensitive [³H]leucine incorporation into protein (due to mitochondrial protein synthesis) in synchronized HeLa cells has been found to continue throughout interphase, its rate per cell approximately doubling from the G₁ to the G₂ phase. This increase in the rate of [³H]leucine incorporation during the cycle does not seem to parallel closely the increase in cell mass. In fact, the observations made on cultures incubated at 34.5 °C, where the G₁ and S phases are better resolved than at 37 °C, indicate that the rate remains constant during the G₁ phase, and starts to accelerate with the onset of nuclear DNA synthesis. Correspondingly, on a per unit mass basis, there appears to be a slight decline in the rate of [³H]leucine incorporation into protein during the G₁ phase, which is compensated by an increase in the early S phase. No significant variations were observed in the mitochondrial leucine pool labeling during the cell cycle; therefore, the observed pattern of [³H]leucine incorporation into protein should reflect fairly accurately the behavior of mitochondrial protein synthesis. Evidence has been obtained indicating a depression in the rate of incorporation of [³H]leucine into protein in mitochondria of mitotic cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the products of mitochondrial protein synthesis has not revealed any differences in the size distribution of the proteins synthesized in the various portions of the cell cycle.     

Influence of collagen gel substratum on response to low-density lipoprotein by cultured human skin fibroblasts

The effect of low-density lipoprotein (LDL) on accumulation of glycosaminoglycans (GAG) was compared in cultures of human skin fibroblasts on a conventional plastic substratum and in a native type I collagen gel. The 24-h incorporation of [3H]glucosamine and Na2(35)SO4 into GAG secreted into the medium or associated with the substratum and cell surface (SCA) was measured in cells at subconfluent densities. When cells were grown on plastic, 13-25% of the labeled GAG was in the SCA pool. Cells cultured within a collagen gel matrix incorporated three times more [3H]glucosamine and up to five times more [35S]sulfate into this pool. The addition of LDL (300 micrograms protein/mL) to the medium increased the level of total GAG incorporation of [3H]glucosamine by 40-50% and of [35S]sulfate by 15-20% on both substrata. For cells on plastic the relative increase in the medium and SCA pool was similar, whereas for cells in collagen gel the response to LDL was twice as great in the SCA pool as in the medium. The distribution of GAG types was unaffected by LDL; hyaluronic acid remained the principal GAG in the media pools of both substrata, heparan sulfate remained the main SCA GAG in cultures on plastic, and dermatan sulfate remained the dominant GAG in the SCA pool of collagen gel cultures. LDL degradation was measured at intervals up to 48 h after the addition of 125I-labeled LDL. The rate of accumulation of degraded LDL products was lower in collagen gel cultures, but the final levels achieved were the same in the two substrata. Concentrations of total cell cholesterol were similar, although the increases in free cholesterol induced by LDL were 26% greater in cells within collagen gel than in those on plastic. We conclude that fibroblasts grown within a collagen gel, as compared with those on a plastic substratum, (i) accumulate more GAG that remain attached to the substratum and cell surface; (ii) respond to LDL with a similar degree of increase in GAG accumulation, but more of the increase is found in the substratum and cell surface compartment; and (iii) accumulate more intracellular free cholesterol in response to LDL.     

Effect of retinoic acid on in vitro proliferation activity and glycosaminoglycan synthesis of mesenchymal cells from palatal shelves of mouse fetuses

To clarify the mechanism by which retinoid causes cleft palate, we investigated the effect of retinoic acid (RA) on proliferation activity and glycosaminoglycan (GAG) synthesis in mouse fetuses palatal mesenchymal (MFPM) cells. MFPM cells were incubated for 1-11 days with various concentrations of RA to examine its effect on growth rate. Also, confluent cultures were incubated with [3H]glucosamine or [35S]sulfate in the presence of various concentrations of RA to investigate the effect of RA on GAG synthesis. RA remarkably inhibited the growth of MFPM cells in a dose-dependent manner. RA also inhibited the synthesis of GAGs, with sulfated GAGs being more severely affected than hyaluronic acid. These data suggest that the inhibition of proliferation activity and GAG synthesis of palatal mesenchymal cells might be involved in the induction of cleft palate by retinoic acid.