Depression by hyaluronic acid of glycosaminoglycan synthesis by cultured chick embryo chondrocytes

Proteins synthesized during the preimplantation period of mouse embryogenesis were labeled with radioactive tyrosine and lysine and fractionated by electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate. For interstage comparisons and comparisons of the incorporation of different amino acids at the same developmental stages, the embryos were incubated with either ³H- or ¹⁴C-labeled amino acids. The embryos were then combined, and the proteins were isolated and electrophoresed simultaneously. The data were analyzed with a dual isotope computer program and expressed in the form of ${}^{14}\text{C}{}^3\text{H}$ ratios.Approximately 20–25 labeled protein components of apparent molecular weights between 25,000 and 115,000 can be defined, and 5 are most significant quantitatively. Of the latter, there are developmental increases in the rates of synthesis of 3 (with apparent molecular weights of 35,000 to 37,000, 37,000 to 41,000, and 66,000 to 70,000), a decrease in the rate of synthesis of another (53,000 to 57,000), and little change in the last (46,000 to 49,000). Developmental changes in the rates of synthesis of several other components are also demonstrated by the ${}^{14}\text{C}{}^3\text{H}$ incorporation ratios. The relative amounts of the different proteins synthesized by day 3 (early blastocyst) embryos over an 8-hr period remain constant, as does the relative labeling by lysine and tyrosine at each developmental stage examined. Similarly, there is no change in the pattern of the radioactive proteins when day 2 (8–16 cell) embryos are labeled for 2 hr and then incubated for an additional 24 hr. The greatest change in the overall pattern of protein synthesis occurs quite early, between day 1 (2 cell) and day 2, and lesser changes occur at later stages. These findings are in contrast to the major change in the rate of protein synthesis which occurs after day 2.     

Radioactive labeling of proteins in cultured postimplantation mouse embryos II. Dose and time dependency

Summary The conditions for optimum incorporation of radioactive amino acids into proteins of cultured postimplantation mouse embryos were investigated under the aspect of using these proteins for two-dimensional electrophoretic separations and fluorography. The aim was to obtain highly radioactively labeled proteins under conditions as physiological as possible. Mouse embryos of Days 8, 10, and 11 of gestation were cultured in Tyrode’s solution. Incubation time and concentration of [³H (or¹⁴C)]amino acids in the culture medium were varied over a broad range. Embryos were prepared with placenta and yolk sac or without any embryonic envelopes. After culturing, the physiologic-morphologic state of the embryos was registered on the basis of several criteria. The radioactivity taken up by the total protein of each embryo was determined and calculated in disintegrations per minute per milligram protein per embryo. To approach our aim, embryos of different developmental stages had to be cultured under different conditions. A good compromise for Day-8, Day-10, and Day-11 embryos was: embryos prepared with yolk sac (opened) and placenta, 150 μCi radioactive amino acids added per milliliter medium, incubation for 4 to 5 h. For maximum labeling of proteins it is advisable to culture Day-10 embryos without embryonic envelopes under particular conditions. This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to the project K1 237/3-2 (Systematic analysis of cell proteins).     

Rates of ribosomal protein and total protein synthesis during Drosophila early embryogenesis

Embryos at various stages of early development from 1.5 to 5 hr after oviposition were made permeable with octane and labeled for 1 hr with [³H]phenylalanine. Measurements of the rate of incorporation of [³H]phenylalanine into ribosomal proteins and total protein were made using these synchronized Drosophila embryos. The rate of synthesis of those ribosomal proteins incorporated into ribosomes increases until 3 to 4 hr after fertilization (550 pg/embryo-hr) then declines later in embryonic development. The rate of total protein synthesis is maximal as early during embryonic development as could be measured. During the period between 1.5 and 2.5 hr after fertilization this rate is 9.4 ng/embryo-hr and then also declines. The synthesis of ribosomal proteins accounts for a substantial portion (4.5%–8.9%) of total protein synthesis in early embryos. These results indicate that ribosome formation is a significant activity during the earliest stages of Drosophila development.     

Ɛ-Fructoselysine as an Absorption-delayed Material in the Small Intestinal Lumen of Rats Fed Browned Casein

Investigations were carried out in order to elucidate the reason for the nutritive value reduction of browned protein by using casein labeled with U-¹⁴C-L-lysine. When the browned casein was ingested by growing rats, high radioactivity was found in a lysine derivative present in the small intestinal TCA-soluble fraction obtained 3 and 7 hr after feeding. Experiments were done to identify the lysine derivative as absorption-delayed material. This material was identified by an amino acid auto-analyzer, paper chromatography and radioactive analysis as l-deoxy-l-(?-N-L-Iysino)-D-fructose (?-fructoselysine), which accounted for about 70% of the total radioactivity in the small intestinal TCA-soluble fraction obtained 7 hr after feeding. Lysine which was found after 3 hr, was disappeared from the intestinal TCA-soluble fraction taken 7 hr after feeding, but e-fructoselysine remainded. Its content as acetate found 7 hr after one-dosal feeding of 600 mg browned labeled casein (700,000 dpm) was 18.3 and 19.0 mg/each rat, respectively. These values accounted for about 40% of the lysine content in the ingested browned casein.     

Changes in synthesis of specific proteins following axotomy: Detection with two-dimensional gel electrophoresis

Changes in protein synthesis during development and following axotomy were analyzed by two-dimensional gel electrophoresis. The two major postganglionic nerves emerging from the superior cervical sympathetic ganglia (SCSG) of adult rats were either cut or crushed unilaterally. At intervals ranging from 1 to 112 days after surgery both SCSG were removed and incubated for 1 hr in the presence of ¹⁴C-leucine. Proteins were extracted and subjected to two-dimensional electrophoretic separation and autoradiography. With this technique, proteins are separated on the basis of isoelectric point and molecular weight. Also, intact SCSG from 1, 2, 7, and 14 day old rats were labeled and analyzed. It was found that a minority of the separated proteins exhibited some detectable change in relative rate of synthesis following axotomy. Actin exhibited a slight (< 20%) increase in relative synthesis rate while tubulin did not change significantly. There were small but significant differences in the protein patterns following nerve crush, as opposed to nerve cut. Comparison of protein synthesis patterns from developing rat SCSG with those from intact and from axotomized adult SCSG failed to demonstrate any marked similarity between the developmental and the axotomy patterns.     

Aspartate kinase and the synthesis of aspartate-derived amino acids in wheat

Aspartate kinase (EC 2.7.2.4.) has been purified from 7 day etiolated wheat (Triticum aestivum L. var. Maris Freeman) seedlings and from embryos imbibed for 8 h. The enzyme was 50% inhibited by 0.25 mM lysine. In this study wheat aspartate kinase was not inhibited by threonine alone or cooperatively with lysine; these results contrast with those published previously. In vivo regulation of the synthesis of aspartate-derived amino acids was examined by feeding [¹⁴C]acetate and [³⁵S]sulphate to 2–3 day germinating wheat embryos in culture in the presence of exogenous amino acids. Lysine (1 mM) inhibited lysine synthesis by 86%. Threonine (1 mM) inhibited threonine synthesis by 79%. Lysine (1 mM) plus threonine (1 mM) inhibited threonine synthesis by 97%. Methionine synthesis was relatively unaffected by these amino acids, suggesting that there are important regulatory sites other than aspartate kinase and homoserine dehydrogenase. [³⁵S]sulphate incorporation into methionine was inhibited 50% by lysine (2 mM) plus threonine (2 mM) correlating with the reported 50% inhibition of growth by these amino acids in this system. The synergistic inhibition of growth, methionine synthesis and threonine synthesis by lysine plus threonine is discussed in terms of lysine inhibition of aspartate kinase and threonine inhibition of homoserine dehydrogenase.Abbreviations AEC S-(2-aminoethyl) cysteine     

Polar lobe specific regulation of translation in embryos of Ilyanassa obsoleta.

Comparisons of the patterns of synthesis of ¹⁴C-labeled proteins of normal embryos of Ilyanassa with the patterns of synthesis of ³H-labeled proteins of embryos from which the polar lobes had been removed at the trefoil stage were made by coelectrophoresis on polyacrylamide disc gels. Controls utilizing biochemical and morphological markers were performed to assure that normal and delobed embryos were developing at equivalent rates. The expression of significant differences in the patterns of protein synthesis were found between normal and delobed embryos, and these differences were not dependent upon concomitant RNA synthesis. These differences were observable as early as after only 24 hr of development, although organogenesis does not begin until much later in development. Therefore, the observed differences probably reflect determininative events. The results support the hypothesis that the developmental determinants of the polar lobe may include specific, preformed mRNA sequences, or specific regulators of translation.     

DNA synthesis in developing two-cell mouse embryos

Mated CF1 (Carworth) female mice were sacrificed at 2 hr intervals between 29 and 43 hr after human chorionic gonadotrophin (HCG) administration. One- and two-cell eggs were incubated in [³H]thymidine for 1 hr. Labeled two-cell embryos were first observed at 31 hr and reached a maximum number at 35 hr. The S period is approximately 6 hr in duration. Although both blastomeres were labeled in most cases, embryos with only one labeled blastomere were more numerous at later times. In vitro labeling was corroborated by injecting [³H]thymidine directly into the isthmic portion of the oviduct. Embryos usually complete the second cleavage division 18–20 hr after onset of DNA synthesis. The cell cycle at the two-cell stage is thus characterized by a G₁ of close to 1 hr, a 6 hr S, and a G₂ of about 12 hr.Embryos developing in vitro frequently fail to progress beyond the two-cell stage. The block is not due to absence of DNA synthesis since these embryos were found to incorporate [³H]thymidine.     

DIURNAL VARIATION IN THE LABELING INDEX OF MOUSE EPIDERMIS: A DOUBLE ISOTOPE AUTORADIOGRAPHIC DEMONSTRATION OF CHANGING FLOW RATES

A diurnal rhythmicity in the labeling index was observed in the epidermis of hairless mice, injected with either ¹⁴C- or ³H-thymidine, at different times during a 24 hr period. A modified autoradiographic technique, using ¹⁴C- and ³H-thymidine and two overlying emulsion layers, makes it possible to clearly differentiate synthesizing cells which are singly labeled with either carbon-14 or tritium, and cells labeled with both isotopes. At various times during a 24 hr period, hairless mice were injected with thymidine-2-¹⁴C and colcemid, followed at 2 or 3 hr by a second injection of ³H-thymidine. The labeling indices were calculated for the ¹⁴C- and ³H-thymidine injection times. These labeling indices were consistent with the control, single isotope, labeling indices and exhibited the same diurnal rhythm. Cells singly labeled with ³H- or ¹⁴C-thymidine have either started or completed DNA synthesis during the interval between the two injections. Flow rates into and out of DNA synthesis, throughout the 24 hr period, can be calculated from these singly labeled cells. The flow rates varied rhythmically throughout the day and paralleled changes in the labeling indices. The influx and efflux flow rates, at all times measured, were not equal. The influx flow rate was reflected in the efflux rate at a time later equal to the duration of S. By means of these flow rates, the per cent of cells in DNA synthesis was calculated for each hour during a 24 hr period. The resulting labeling index curve matches the observed 24 hr diurnal rhythm in labeling indices. By extension of these flow rates through mitosis, the resulting mitotic index curve is comparable to the reported 24 hr diurnal rhythm in mitotic indices.     

Preparation of radioactive epsilon-N-methylated lysines not involving elaborate organic synthesis.

A simple method to prepare small quantities of radioactive ε-N-methylated lysines for use as analytical standard markers, which does not involve elaborate organic synthesis, is highly desired in numerous research applications. The method in this report involved growingNeurospora crassa orSalmonella typhimurium in the presence of uniformly labeled [U-¹⁴C] lysine or reductively methylating a protein with [¹⁴C]formaldehyde at pH 9.0. The radiolabeled proteins were then isolated and hydrolyzed in 6n HCl. Finally, the radioactive methylated lysines in the acid hydrolysates were isolated by Dowex 50 column chromatography.     

Rates of synthesis of major classes of RNA in Drosophila embryos.

We have been successful in labeling to high specific activity (3 × 10⁵ dpm/μg) the RNA synthesized by large numbers of Drosophila embryos. Embryos of various developmental stages were rendered permeable with octane and labeled with [³H]uridine for 1 hr. At each stage the total dpm incorporated into RNA and the specific activity of the UTP pool were measured and used to calculate the absolute rate of RNA synthesis per embryo. This rate increases during embryonic development, from 1 pmole UTP/hr at 2 hr after oviposition to 6 pmoles UTP/hr at 15 hr. The rates of synthesis of nuclear and cytoplasmic poly(A)^? and poly(A)⁺ RNAs were determined by analyzing the fractionated RNAs from each stage by sucrose gradient sedimentation. There is a significant activation of nuclear RNA synthesis at the blastoderm stage (approximately 2 hr after oviposition). After blastoderm, the rates of synthesis of nuclear and cytoplasmic poly(A)^? and poly(A)⁺ RNA per embryo increase continuously; the rate of synthesis of each of these classes per nucleus, however, remains fairly constant. After making corrections for turnover during the labeling period, we find that the rates of synthesis of the major classes of RNA per nucleus at the gastrula stage are: cytoplasmic poly(A)⁺ RNA, 0.06 fg/nucleus-min; hnRNA, 0.86 fg/nucleus-min; and ribosomal RNA, 0.46 fg/nucleus-min. These rates are compared to rates of RNA synthesis in sea urchin embryos.     

Chimeras vs X inactivation mosaics: significance of differences in pigment distribution

Cell cultures of cardiac, pectoral, and thigh muscle of chick embryos synthesized myoglobin, as measured by incorporation of radioactive lysine detected by radioimmunoprecipitation. Liver and skin cultures, although active in protein synthesis, failed to demonstrate myoglobin synthesis. Puromycin inhibited myoglobin synthesis by the cell cultures. The electrophoretic characteristics of the myoglobin antigen synthesized by thigh and pectoral muscle were identical. Myglobin synthesizing progenitor cells attached to plastic dishes in 1 hr, but not completely in 0.5 hr. Cells, unattached at 0.5 hr, were enriched in myoglobin synthesizing cells. Incorporation of lysine-U-¹⁴C into myoglobin was maximal in confluent cultures and its increase paralleled the increase of cell fusion in the cultures. The ability of pectoral, white muscle to synthesize myoglobin in a manner equivalent to that of cardiac tissue was unexpected because of its failure to synthesize myoglobin in vivo and may indicate that factors in the whole organism may regulate the expression of this muscle cell's capabilities.     

Plasmodium falciparum: Synthesis of glycoprotein by cultured erythrocytic stages

The human malarial parasite, Plasmodium falciparum, incorporated significant radioactivity into glycoconjugates when cultured in the presence of [¹⁴C]- or [³H]glucosamine for 48 to 50 hr. Digestion of the labeled proteins with pronase and subsequent precipitation with absolute ethanol showed that 90 to 95% of the radioactive glucosamine was incorporated into the precipitated material. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the labeled macromolecules revealed eight bands with approximate molecular weights from 19,000 to 90,000 daltons.     

Hypusine, N6-(4-amino-2-hydroxybutyl)-2,6-diaminohexanoic acid,in tissue proteins of mammals

Hypusine, N⁶-(4-amino-2-hydroxybutyl)-2,6-diaminohexanoic acid was isolated from proteins of bovine brain. Its identification was performed by comparison of its behavior in amino acid analysis, paper chromatography and electrophoresis to that of the authentic compound, and by periodic acid-permanganate oxidation which split hypusine into β-alanine and lysine. Hypusine was found in proteins of various organs of rabbits.Formation of hypusine from lysine was demonstrated by the intraperitoneal injection of labeled lysine into a rat and isolation of radioactive hypusine from the animal proteins. This findings indicates a possibility that hypusine is derived from the lysine residue of proteins through attachment of the 4-amino-2-hydroxybutyl moiety to the N⁶-amino radical of lysine.     

Biosynthesis and secretion of tropoelastin by chick aorta cells

Cells were isolated from the aortae of 17-day old chick embryos by digestion of the vessels with a combination of trypsin and collagenase. When these cells were incubated in suspension culture in Krebs-Ringer media containing pancreatic trypsin inhibitor and radioactive amino acids, they synthesized and secreted labeled proteins into the media. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the secreted proteins labeled with [¹⁴C]proline revealed two major components. The larger component with an approximate molecular weight of 125,000 had a [¹⁴C]hydroxyproline content consistent with a form of procollagen. The molecular weight of 70,000 and [¹⁴C]hydroxyproline content of the second component was consistent with that previously reported for tropoelastin extracted from chick aortae. By following the kinetics and secretion of tropoelastin labeled with [³H]valine, we have estimated that 17 minutes are required to synthesize and secrete the molecule under these experimental conditions.     

Induction of the primitive streak in the chick blastoderm embryo: patterns of protein synthesis

Summary Induction of the primitive streak is correlated with specific qualitative and quantitative changes in protein synthesis in the component areas of chick blastoderm. Blastoderm embryos at the initial to intermediate primitive streak stage were labeled with L-[³⁵S] methionine. Radioactively labeled proteins separated by two-dimensional sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis revealed differences in the number and density of spots among the component areas of the epiblast and hypoblast. Protein patterns of the area opaca, marginal zone and central area of the epiblast are very similar qualitatively but show distinct quantitative differences. A comparison between any of the component areas of the epiblast and the hypoblast in chick blastoderm embryos, however, reveals both qualitative and quantitative differences. A protein with a molecular weight of 30,000 unique to the component areas of the epiblast, and proteins with a molecular weight of 22,000 and 37,000 unique to the hypoblast are prominent and seem to be related to the initial appearance of the primitive streak.     

Rates of histone synthesis during early development of Rana pipiens

Newly synthesized histones have been extracted from Rana pipiens oocytes or cleaving embryos previously injected with [³H]lysine or [³H]arginine. The radioactive proteins were fractionated by cation-exchange chromatography and electrophoresis on acid/urea or SDS-polyacrylamide gels; histones were identified by coelectrophoresis with authentic markers. From percentage total incorporation in the putative histones, and absolute rates of lysine or arginine incorporation, rates of histone synthesis were estimated. Rates of histone synthesis in two-cell embryos were at least 10-fold higher than in maturing oocytes. Between the two-cell and blastula stages, the rate increased an additional threefold, from about 1200 pg hr^?1 per embryo to about 4500 pg hr^?1 per embryo. While all histone classes are synthesized during cleavage, synthesis of the various classes is not coordinated; histones are not synthesized in the same relative proportions at which they are found in blastula chromatin. The synthesis of histone H4 in particular is barely detectable during cleavage. This, and other observations, suggested the existence of cytoplasmic histone pools. In approaching the possible existence of histone pools, the amount of H4 present in oocytes was determined. Oocytes contain about 74 ng of H4, an amount sufficient to allow development to the blastula stage. These data are compared to those reported by others on histone synthesis during cleavage in Xenopus.     

Size and specific activity of the UTP pool and overall rates of RNA synthesis in early mouse embryos

Mouse embryos from the one-cell to the blastocyst stage were cultured for 2 hr in the presence of 5 μM [³H]uridine or 10 μM [³H]adenosine, and the size and specific activity of the UTP and ATP pools were determined by an Escherichia coli RNA polymerase assay using synthetic poly(dA-dT) as template. The total UTP pool increased in size and specific activity with development from 0.05 pmole (0.06% labeled) in the one-cell stage to 0.54 pmole (27% labeled) in the blastocyst stage. The total ATP pool remained relatively constant in size at about 1 pmole/embryo, but increased in specific activity from 2.6 to 52% from one-cell to blastocyst. The turnover of the [³H]UTP pool was also examined under pulse-chase conditions in eight-cell and morula-stage embryos. The UTP pool decayed with approximately first-order kinetics up to 20 hr of chase, but the rate of decay was slower in eight-cell embryos (t_0.5 = 5.5 hr) than in morulae (t_0.5 = 2.8 hr). The observed specific activities of the UTP pools were used to calculate the overall rates of uridine incorporation into acid-precipitable material during early development. The rate of uridine incorporation per embryo increased from 3.6 × 10^?3 pmole/2 hr in the two-cell embryo to 1.8 × 10^?1 pmole/2 hr in the blastocyst. The rate of RNA synthesis per cell over a 2-hr period was estimated at 2.5 pg in the two- to four-cell embryo, 5 pg in the eight-cell, and 10 pg in the morula-early blastocyst.     

Renal glomerular collagen synthesis in streptozotocin diabetes Reversal of increased basement membrane synthesis with insulin therapy

The effect of diabetes and insulin on basement membrane synthesis in vitro be renal glomeruli obtained from normal and diabetic rats was determined. Four groups of experimental animals were used: age-matched controls; streptozotocin-diabetic; and streptozotocin-diabetic treated with insulin for half or all of the duration of diabetes. Isolated glomeruli were incubated with [¹⁴C]-lysine and the radioactive lysine and hydroxylysine in glomerular proteins were measured. [¹⁴C]Lysine incorporation and hydroxy[¹⁴H]lysine synthesis were elevated in diabetic glomeruli. Progressive diminution in ¹⁴C-labelled protein and hydroxy[¹⁴C]lysine formation was observed in incubations containing glomeruli from insulin-treated diabetic rats, with greater reversal toward normal following longer periods of exogenous insulin administration. Basement membrane synthesis, determined by the appearance of labelled hydroxylysine in membranes obtained from sonicated glomeruli, was increased in diabetic preparations. Reversal of these changes toward normal values was observed in glomeruli from rats treated with insulin immediately following induction of diabetes. The results indicate that basement membrane synthesis is increased in renal glomeruli from streptozotocin-diabetic rats, and that this process is restored toward normal with continuous insulin therapy.