THE EFFECT OF EXTRACELLULAR POTASSIUM CONCENTRATION ON PROTEIN SYNTHESIS IN GUINEA-PIG HIPPOCAMPAL SLICES1,2

Abstract— We have measured the effect of small variations in extracellular potassium concentrations ([K⁺]) upon the incorporation of radioactively labelled amino acid into the protein of the isolated guinea-pig hippocampal slice. The slice is super-perfused with glucose fortified buffer and maintains an ATP concentration of 33–36 nmol/mg protein and incorporates lysine into protein at a rate of 0.82 pmol/(ig protein/h. Within the range of extracellular K⁺ from 1.3 to 8.1 mil the change in the rate of lysine incorporation into protein is proportional to the logarithm of the extracellular K⁺ concentration. Incorporation increases by about 100% over this range. Measurements of the specific activity of the presumed intracellular amino acid pool indicate that the effect of changes in extracellular [K⁺] is to alter the rate of protein synthesis rather than alter the availability of radioactively labelled precursor. Altering extracellular [K⁺] does not affect tissue levels of ATP or creatine phosphate, indicating that the effect on amino acid incorporation does not result from an effect upon energy metabolism. It is suggested that this effect of extracellular [K.⁺] may be a means by which changes in cerebral electrical activity lead to changes in the rate of protein synthesis in brain.     

EXTRACELLULAR STIMULATION OF EPIDERMAL DNA SYNTHESIS

The removal of the topmost cell layers of the epidermal stratum corneum by stripping initiates a series of biochemical events which alters the normal homeostatic control of and results in the acceleration of the cell cycle in basal cells which are ten to twenty cell layers removed from the site of stripping. One measure of accelerated events is a stimulation of thymidine incorporation into epidermal DNA at time intervals following stripping. Two peaks of maximal stimulation occur between 12 and 24 hr and 48 and 54 hr after stripping. Stimulation of thymidine incorporation into epidermal DNA by limited stripping is a useful technique for studying the stripping-initiated signal at the stratum corneum and its subsequent translation at the proliferative cell receptor site.     

IN VITRO STIMULATION OF ENZYME SECRETION AND THE SYNTHESIS OF MICROSOMAL MEMBRANES IN THE PANCREAS OF THE GUINEA PIG

Several mechanisms have been suggested to explain how secretory cells remove from the plasmalemma the excess membrane resulting from the insertion of granule membrane during exocytosis: intact patches of membrane may be internalized and then reutilized within the cell; alternatively these membranes may be either disassembled to subunits or degraded. In the latter case new membranes should be synthetized at other sites of the cell, probably in the rough-surfaced endoplasmic reticulum (RER) and the Golgi complex. In the present research, membrane subfractions were obtained from rough microsomes (derived from fragmented and resealed RER cisternae) and from smooth microsomes (primarily contributed by Golgi stacks and vesicles) of the guinea pig pancreas by incubation at 4°C for 4 hr in 0.0005 M puromycin at high ionic strength followed by mild (pH 7.8) alkaline extraction with 0.2 M NaHCO₃. Such treatments release the majority of nonmembrane components of both microsomal fractions (i.e., contained secretory enzymes, ribosomes, and absorbed proteins of the cell sap) and allow the membranes to be recovered by centrifugation. The effect of in vitro stimulation of enzyme secretion (brought about in pancreas slices by 0.0001 M carbamoyl choline) on the rate of synthesis of the phospholipid (PLP) and protein of these membranes was then investigated. In agreement with previous data, we observed that in stimulated slices the synthesis of microsomal PLP was greatly increased. In contrast, the synthesis of microsomal membrane proteins was unchanged. These results suggest that exocytosis is not coupled with an increased rate of synthesis of complete ER and Golgi membranes and are, therefore, consistent with the view that excess plasma membrane is preserved and reutilized, either as discrete membrane patches or as membrane macromolecules, throughout the secretory cycle.     

PROTEIN SYNTHESIS IN VITRO IN NUCLEI OF HUMAN NERVOUS TISSUE

Abstract—

• 1 In vitro incorporation of tritiated leucine into nuclear proteins of normal human brain, astrocytoma I and II and glioblastoma multiforme was investigated. The distribution of radioactivity among various protein fractions of nuclei was determined.
• 2 The results demonstrate that the isolated nuclei of astrocytoma I and II incorporate radioactivity into proteins at least 40 times more actively than nuclei of normal human brain or glioblastoma multiforme.
• 3 The residual protein fraction was the most highly labelled fraction among the nuclear pioteins. This fraction from astrocytomas incorporates relatively more radioactivity than the similar fraction of normal human brain or glioblastoma multiforme. The buffered-saline soluble protein fraction of astrocytoma nuclei contained a relatively lower amount of radioactivity than the similar fraction of normal human brain or glioblastoma multiforme. The total radioactivity incorporated into the deoxyribonucleoproteins seemed to increase with the malignancy of the tissue investigated. The significance of the results with respect to malignant transformation is discussed.

     

THE EFFECTS OF PHENYLALANINE ON AMINO ACID METABOLISM AND PROTEIN SYNTHESIS IN BRAIN CELLS IN VITRO1

Incubation of brain cell suspensions with 14 mM-phenylalanine resulted in rapid alterations of amino acid metabolism and protein synthesis. Both thc rate of uptake and the final intracellular concentration of several radioactively-labelled amino acids were decreased by high concentrations oi phenylalanine. By prelabelling cells with radioactive amino acids, phenylalanine was also shown to effect a rapid loss of the labelled amino acids from brain cells. Amino acid analysis after the incubation of the cells with phenylalanine indicated that several amino acids were decreased in their intracellular concentrations with effects similar to those measured with radioisotopic experiments (large neutral > small and large basic > small neutral > acidic amino acids). Although amino acid uptake and efflux were altered by the presence of 14 mwphenylalanine, little or no alteration was detected in the resulting specific activity of the intracellular amino acids. High levels of phenylalanine did not significantly altcr cellular catabolism of either alanine, lysine, leucine or isoleucine. As determined by the isolation of labcllcd aminoacyl-tRNA from cells incubated with and without phenylalanine, there was little or no alteration in the level of this precursor for radioactive alanine and lysine. There was, however, a detectable decrease in thc labelling of aminoacyl-tRNA for leucine and isoleucine. Only aftcr correcting for the changes of the specific activity of the precursors and thcir availability to translational events, could the effects of phenylalanine on protein synthesis be established. An inhibition of the incorporation into protein for each amino acid was approximately 20%.     

ON THE MECHANISM OF ELECTROSHOCK-INDUCED INHIBITION OF PROTEIN SYNTHESIS IN RABBIT CEREBRAL CORTEX

Abstract— The mechanism of electroshock (ES)-induced inhibition of protein synthesis in rabbit cerebral cortex has been investigated by using a cell-free system. The protein biosynthetic activity of the post-mitochondrial supernatant (PMS) obtained from the cerebral cortex of ES-treated animals was found to be markedly lower than in controls (C). This inhibition was accompanied by a decrease of polysomes and an increase of monomers. In addition, a relative increase in light polysomes was evident at short intervals after ES treatment. No difference was found in the total soluble activity and in the activity of the elongation factors and ribonuclease present in the cell sap of C and ES animals. The biosynthetic activity of ES-total. free and membrane-bound ribosomes was approx 45% lower than that of the corresponding C fractions: polysome/monomer ratios were similarly reduced. The total content of cortical ribosomes was not affected by ES. Following ES treatment there was no change in the ribo-somal ability to elongate, terminate and release polypeptide chains, nor a decrease in the polysomal content of poly(A)-containing mRNA. These data strongly suggest that the ES-induced inhibition of protein synthesis results from a defect in the initiation process. The possible mechanisms mediating this defect have been discussed.     

ON THE MECHANISM OF OUABAIN INHIBITION OF SYNAPTOSOME PROTEIN SYNTHESIS

Abstract— Ouabain (200μ m ) inhibited incorporation of radiolabelled leucine or glycine into the protein of neonatal synaptosome fractions but had minimal effect on preparations from adult rats. Leucine uptake into synaptosomes was rapid but not influenced by 200μ m -ouabain in contrast to ouabain inhibition of [¹⁴C]glycine and [¹⁴C]γ-aminobutyric acid uptake. Ouabain blocked the Na⁺ -dependent (stimulated) component of synaptosome fraction protein synthesis in the presence of 25m m -K⁺. Ouabain inhibition was not alleviated by addition of ADP or ATP. 100μ m -atractylate failed to influence [³H]leucine uptake or incorporation. Synergistic inhibition by ouabain was observed with the cycloheximide-sensitive component of protein synthesis and the chloramphenicol sensitive phase. Increasing the medium Ca²⁺ concentration stimulated protein synthesis and this stimulated component was inhibited by ouabain. Ouabain inhibition was associated with decreasing intraterminal K⁺ concentration and [K]_i was linearly related to the protein synthesis rate in control and ouabain treated preparations.     

INFLUENCE OF EXTERNAL POTASSIUM ON THE SYNTHESIS AND DEPOSITION OF MATRIX COMPONENTS BY CHONDROCYTES IN VITRO

The effect of a high external potassium concentration on the synthesis and deposition of matrix components by chondrocytes in cell culture was determined. There is a twofold increase in the amount of chondroitin 4- and 6-sulfate accumulated by chondrocytes grown in medium containing a high potassium concentration. There is also a comparable increase in the production of other sulfated glycosaminoglycans (GAG) including heparan sulfate and uncharacterized glycoprotein components. The twofold greater accumulation of GAG in the high potassium medium is primarily the result of a decrease in their rate of degradation. In spite of this increased accumulation of GAG, the cells in high potassium fail to elaborate appreciable quantities of visible matrix, although they do retain the typical chondrocytic polygonal morphology. Although most of the products are secreted into the culture medium in the high potassium environment, the cell layer retains the same amount of glycosaminoglycan as the control cultures. The inability of chondrocytes grown in high potassium to elaborate the typical hyaline cartilage matrix is not a consequence of an impairment in collagen synthesis, since there is no difference in the total amount of collagen synthesized by high potassium or control cultures. There is, however, a slight increase in the proportion of collagen that is secreted into the medium by chondrocytes in high potassium. Synthesis of the predominant cartilage matrix molecules is not sufficient in itself to ensure that these molecules will be assembled into a hyaline matrix.     

DEVELOPMENTAL EFFECTS ON PROTEIN SYNTHESIS RATES IN REGIONS OF THE CNS IN VIVO AND IN VITRO

Abstract— Protein synthesis rates have been determined quantitatively in several regions of the nervous system of rats of various ages. The developmental changes in these regions are generally similar with a high rate maintained from several days before birth to about 4 days of age (1.9–2.1% h⁻¹). A decline in the rate ensues thereupon which continues till approx 30 days of age, whence the curve flattens though continuing slowly downward with increasing age. In the young three regions, cerebellum, pineal and pituitary, exhibit exceptionally higher rates (40–50%) than the cerebral hemispheres, pons-medulla, mid brain or cord, which all display curves of similar magnitude and shape. While the rate in the cerebellum eventually declines with age to within 10% of the rate in cerebral hemisphere, rates in the pineal and pituitary though decreasing remain far above (100%) rates in cerebral hemisphere even in adults.
The rate in vitro for slices of cerebellum follows a pattern similar to that shown previously for cerebral hemispheres: in the very young rates are 70–80% of the in vivo value but decline much more rapidly with age and in adult represent only 10–15% of the rate in vivo.
A markedly different pattern is seen in whole (unsliced) pituitaries wherein in vitro rates parallel in vivo rates with increasing age at approx 70–80% of the in vivo rate. Pineals appear to follow a similar pattern.     

THE SUBSTRATE IN PEPTIC SYNTHESIS OF PROTEIN

1. Experiments are described in which it was observed that the yield of protein that can be synthesized by pepsin from a given peptic digest is highest when the hydrolyzing action of the pepsin is stopped as soon as all the protein has disappeared from the solution; and that the longer the digest is permitted to contain active enzyme the more the yield diminishes. 2. Exposure of the digest to a hydrogen ion concentration of pH 1.6 in the absence of active enzyme, does not cause a diminution in the amount of protein which can be synthesized from that digest. 3. Synthesis can be effected also in concentrated solutions of isolated fractions of a peptic digest, i.e. of proteose and of peptone. The yields are approximately the same as in similar concentrations of the whole digest, though the proteins so synthesized differ in some respects from those obtained from the whole digest. 4. The cessation of synthesis in any one digest is due to the attainment of equilibrium and not to the complete utilization of available synthesizeable material. The amount of the equilibrium yield, on the other hand, is dependent on the amount of synthesizeable material in the digest. 5. These observations are taken to show that the synthesizeability of a given mixture of protein cleavage products by pepsin depends upon its possession of a special complex in these products. This complex appears as a result of the primary hydrolysis of the protein molecule by pepsin and is decomposed in the slow secondary hydrolysis which ensues as digestion is prolonged.     

STUDIES OF THE MECHANISM OF DEMYELINATION REGIONAL DIFFERENCES IN MYELIN STABILITY IN VITRO1

—Incubation of slices of rat central nervous system in Krebs-Ringer bicarbonate buffer produced a lipoprotein fraction which floated on 10·5% sucrose after homogenization of the slices and centrifugation. This fraction was not found after homogenization and centrifugation of fresh tissue and appeared to depend upon incubation. The amount of the light fraction increased in the following order per 100-mg slice: cerebrum < thalamic area < cerebellum < brain stem < spinal cord. The lipid composition of this fraction was similar to that of myelin, but contained a lower protein content compared to myelin of the corresponding area. This fraction was termed ‘dissociated myelin’. Upon incubation of slices a portion of the basic protein was lost from myelin subsequently isolated, and the dissociated fraction was slightly enriched in basic protein. The distribution of myelin protein among the characteristic three groups (basic, proteolipid and high mol. wt.) was quite different in myelin from spinal cord compared to that from other CNS area. Spinal cord myelin contained about 17% protein compared to about 23% in cerebrum, with brain stem myelin intermediate (19%), and the difference appeared to be due to lesser amounts of proteolipid in the caudal areas. The amount of dissociation after incubation was about 3–5 per cent of the total myelin in the cerebral cortex, 10 per cent in the thalamic area, 20 per cent in cerebellum, 35 per cent in the brain stem, and around 45 per cent in spinal cord. The smaller amount of proteolipid protein in spinal cord myelin may result in a deficiency of cohesive forces holding lipids and proteins together, thus causing greater instability and dissociation. Myelin dissociation increased with time of incubation up to 3 h, was augmented by Ca²⁺, and was substantial at pH 11, reaching a peak at pH 7, then decreased in the acid range. A similar fraction has been isolated previously from fresh CNS tissue made edematous by chronic treatment of rats with triethyl tin. The possible relationship of swelling in the disease process and myelin dissociation are discussed.     

PROTEIN SYNTHESIS IN VITRO IN RAT BRAIN AFTER SHORT-TERM PROTEIN STARVATION AND REFEEDING

Rats were fed a protein-free diet for 4 or 6 days. They were compared with rats kept on the same diet for 3 or 5 days and on adequate protein for one additional day. The incorporation of ¹⁴C-labelled amino acid into protein was studied in systems containing ATP, GTP, phosphoenolpyruvate, pyruvate kinase and if required, a mixture of unlabelled amino acids and either the 6000 g supernatant fraction of a brain homogenate or microsomes and soluble enzymes. The 6000 g supernatant fraction showed variation in amino acid incorporating activity as well as in RNase activity as measured by breakdown of labelled polyuridylic acid. There was no difference in RNase activity in isolated microsomes, but the amino acid incorporating activity was significantly higher in preparations obtained from rats fed one meal of protein after 5 days of protein-starvation.     

ANOMALOUS STIMULATION OF PROTEIN SYNTHESIS: EVIDENCE FOR TRANSLATIONAL CONTROL IN PRIMARY CELLS

Chick cells in primary culture synthesize protein at an accelerated rate following recovery from prolonged (2-12 hr) inhibition of protein synthesis. This 'overshoot' is followed by a return to the normal rate of synthesis.
Both actinomycin and fluorophenylalanine provided during the recovery period enhance the 'overshoot' and prevent the return to normal.
The results are consistent with the proposal that protein synthesis is restricted by an inhibitor or 'governor', itself a protein of relatively short half life.
Neither L nor HeLa cells demonstrate an 'overshoot' during the recovery phase.     

FETAL DEVELOPMENT: THE EFFECTS OF MATURATION ON IN VITRO PROTEIN SYNTHESIS BY MOUSE BRAIN TISSUE

—The elucidation of the translational regulatory events which function during the critical fetal and neonatal period is an important prerequisite to our understanding of normal, as well as abnormal, brain growth and differentiation. Brain cell suspensions and cell-free homogenates were employed to study the protein synthetic activity during the maturation of fetal- neural tissue. The results clearly demonstrated that while neural tissue from 1-day postnatal mice was 10 times more active in protein synthesis than brain tissue from adult mice, the former was many fold less active in translational events than fetal neural tissue from 13-day post-zygotic mice. Fetal polypeptide synthetic activity was found to decrease from the 13th day to the 19th day post-zygotic. This decrement in the translational activity was not due to amino acid availability or pools, or to differences, quantitatively or qualitatively, in polysome concentrations. The enhanced rate of protein synthetic activity measured with neural tissue from 13-day post-zygotic mice was shown to be due to an increase in rate of protein synthesis and not to an enhanced rate of protein degradation.     

LIMITED DNA SYNTHESIS IN THE ABSENCE OF PROTEIN SYNTHESIS IN PHYSARUM POLYCEPHALUM

Actidione (cycloheximide), an antibiotic inhibitor of protein synthesis, blocked the incorporation of leucine and lysine during the S phase of Physarum polycephalum. Actidione added during the early prophase period in which mitosis is blocked totally inhibited the initiation of DNA synthesis. Actidione treatment in late prophase, which permitted mitosis in the absence of protein synthesis, permitted initiation of a round of DNA replication making up between 20 and 30% of the unreplicated nuclear DNA. Actidione treatment during the S phase permitted a round of replication similar to the effect at the beginning of S. The DNA synthesized in the presence of actidione was replicated semiconservatively and was stable through at least the mitosis following antibiotic removal. Experiments in which fluorodeoxyuridine inhibition was followed by thymidine reversal in the presence of actidione suggest that the early rounds of DNA replication must be completed before later rounds are initiated.     

IN VITRO PROTEIN SYNTHESIS ON FREE POLYRIBOSOMES ISOLATED FROM THE DEVELOPING MOUSE BRAIN

Abstract— Free polyribosomes were isolated at three ages (newborn, 2 weeks and 8 weeks) from the developing mouse brain. All three preparations were found to be highly polyribosomal in nature and were essentially identical with respect to their chemical composition and sedimentation properties. An estimate of the sedimentation coefficients of the first seven members of these polysome preparations yielded S °_20,w values of 76, 114, 146, 174, 196, 217 and 236. All three preparations were found to be very active when employed in in vitro protein synthesizing systems. An age-dependent response to the concentration of K⁺ was observed in the activities of the in vitro protein synthesizing systems. Optical K⁺ concentrations for the 0, 2 and 8 week old systems were 30, 50 and 65 mm, respectively. No such age dependence was observed when NH⁺₄ was used as the sole monovalent cation, with all systems exhibiting maximal activity at 50mm-NH⁺₄. The highest in uitro activities were consistently observed (at all three ages) when NNH⁺₄ was employed as the sole monovalent cation. Under optimal conditions, the newborn in vitro protein synthesizing system was observed to be approx 40% as active as either the 2 week or the 8 week systems which were equivalent in activity. The reduced activity of the newborn system appeared to be a function of both the polyribosomal and pH 5 enzyme preparations.     

TIMING OF DNA SYNTHESIS IN THE MITOTIC CYCLE IN VITRO

A study was made of the timing of DNA synthesis in the mitotic cycle under conditions where the average mitotic cycle of populations of human amnion and kitten lung cells in culture was variable. Three types of experiments were performed: (a) Autoradiographs were made of incorporated tritiated thymidine in cells whose mitotic histories were recorded microcinematographically allowing the measurement of telophase + G1 along with the total length of the mitotic cycle. (b) Measurement of the G2 + prophase part of the mitotic cycle was performed under various conditions by exposing cells to tritiated thymidine and observing the increase in labeled metaphases plus anaphases as a function of time. (c) The effect of a change in pH on parts of the mitotic cycle was tested by continuously photographing a single colony of cells first at pH 7.8 and then at pH 7.1. All of our data point to the same conclusion; namely, that within a population of cells with a given generation time, the length of each of the measurable parts of the mitotic cycle has a particular distribution of values and that, when there is a change in the generation time, under our conditions only the T + G1 distribution changes.     

CELL MEMBRANE RESORPTION IN THE RAT EXOCRINE PANCREAS CELL AFTER IN VIVO STIMULATION OF THE SECRETION, AS STUDIED BY IN VITRO INCUBATION WITH EXTRACELLULAR SPACE MARKERS

Pancreatic secretion in the rat was stimulated in vivo by pilocarpine injection causing 90% of the storage granules to be discharged within 2 h. Incubation in vitro with [¹⁴C]sorbitol indicated that maximal ingestion of this extracellular space marker occurred 3 h after secretogogue injection. Morphological cell membrane measurements on cells with stimulated secretion revealed a simultaneous decrease in amount of membrane bordering the microvilli at the cell apex, lamellar processes, and infoldings present at the latero-basal face of these cells. In 3-h stimulated cells, having the average zymogen granule content characteristic for that phase of secretion, ferritin treatment in vitro showed that the infoldings and related fragmentation vesicles had ingested ferritin and could consequently be considered as being transport vehicles for redundant cell membrane. During stimulated secretion numerous vesicles and vacuoles appeared in the apical cytoplasm. Part of these structures were postulated to be related to the Golgi complex and were discussed in relation to secretory protein transport. Another part of these structures was assumed to have an endocytotic nature, although they never contained ferritin.     

植物离体培养中器官发生调控机制的研究进展

本文就离体培养的植物组织对生长调节物质的吸收和代谢，外源生长调节物质对内源激素水平的影响，内源激素对细胞脱分化和再分化的调控，生长素和细胞分裂素基因与器官发生的关系，与器官发生有关的基因和特异蛋白等问题的研究进展进行了评述，并对下一步研究提出了自己的看法。