Amino acid and energy requirements for rat hepatocytes in primary culture

Summary The amino acid and energy requirements of rat hepatocytes in suspension and early culture were investigated. Among a number of potential energy substrates tested, pyruvate (20 mM) was found to be most effective in stimulating hepatocytic protein synthesis. Amino acids stimulated protein synthesis both as energy substrates and as protein precursors. An amino acid mixture was designed to provide maximal inhibition of protein degradation as well as maximal stimulation of protein synthesis. In a defined medium containing amino acids at these concentrations, and supplemented with glucocorticoid hormone and insulin, hepatocytes could be maintained—on a collagen substratum—for at least a week without any significant net loss of cells or cellular protein. The work was supported by grants from The Norwegian Cancer Society and from The Norwegian Council for Science and the Humanities. An erratum to this article is available at .     

Differential regulation of protein synthesis by amino acids and insulin in peripheral and visceral tissues of neonatal pigs

The high efficiency of protein deposition during the neonatal period is driven by high rates of protein synthesis, which are maximally stimulated after feeding. In the current study, we examined the individual roles of amino acids and insulin in the regulation of protein synthesis in peripheral and visceral tissues of the neonate by performing pancreatic glucose–amino acid clamps in overnight-fasted 7-day-old pigs. We infused pigs (n = 8–12/group) with insulin at 0, 10, 22, and 110 ng kg^−0.66 min⁻¹ to achieve ~0, 2, 6 and 30 μU ml⁻¹ insulin so as to simulate below fasting, fasting, intermediate, and fed insulin levels, respectively. At each insulin dose, amino acids were maintained at the fasting or fed level. In conjunction with the highest insulin dose, amino acids were also allowed to fall below the fasting level. Tissue protein synthesis was measured using a flooding dose of l-[4-³H] phenylalanine. Both insulin and amino acids increased fractional rates of protein synthesis in longissimus dorsi, gastrocnemius, masseter, and diaphragm muscles. Insulin, but not amino acids, increased protein synthesis in the skin. Amino acids, but not insulin, increased protein synthesis in the liver, pancreas, spleen, and lung and tended to increase protein synthesis in the jejunum and kidney. Neither insulin nor amino acids altered protein synthesis in the stomach. The results suggest that the stimulation of protein synthesis by feeding in most tissues of the neonate is regulated by the post-prandial rise in amino acids. However, the feeding-induced stimulation of protein synthesis in skeletal muscles is independently mediated by insulin as well as amino acids.     

Mediation of growth hormone-enhanced expression of the cartilage phenotype in vitro by the availability of the essential amino acid valine

Without increasing cell number, ovine growth hormone was shown to stimulate the incorporation of ²⁵SO₄ by cultured chick embryo chondrocytes into chondroitin sulfate. Since the stimulation of sulfation by growth hormone was abolished when the amino acid concentrations in the medium were doubled, the relationship between amino acids and growth hormone in promoting the synthesis of acid mucopolysaccharides was investigated. Comparison of the incorporation of various labeled amino acids into trichloroacetic acid-soluble and insoluble material revealed that growth hormone promoted the incorporation of only valine into trichloroacetic acid-insoluble material. Furthermore, growth hormone stimulated valine incorporation into both extracellular and intracellular protein, rather than preferentially into extracellular chondromucoprotein. Growth hormone gave a 4-fold stimulation of valine incorporation into collagen without stimulating collagen synthesis. That growth hormone enhances sulfation by stimulating valine availability was further supported by the observations: (a) doubling only the valine concentration in the medium enhanced sulfation; (b) in medium with twice the normal valine concentration, sulfation failed to be further stimulated with the addition of growth hormone; and (c) in medium with all the other amino acids except valine at twice normal concentrations, growth hormone enhanced sulfation. In addition the temporal relationships and synthetic events occurring between growth hormonealtered valine availability and enhanced chondromucoprotein synthesis were studied. It was found that growth hormone-promoted valine incorporation into acid-insoluble material is a rapid effect that can be detected by 10 min after hormone addition and does not require RNA synthesis. Increased valine availability is rapidly reversed after growth hormone removal ( ). On the other hand, growth hormone- and valine-enhanced chondromucoprotein synthesis are slower responses, taking over 24 hr of treatment for a maximal stimulation, and are mediated by RNA synthesis, as indicated by actinomycin D sensitivity. Enhanced chondromucoprotein synthesis is also relatively stable after removal of growth hormone or valine ( ).The evidence suggests that the availability of a single amino acid, valine, plays a regulatory role in the synthesis of a specialized cellular product and that growth hormone acts at some level to alter the availability of this essential amino acid.     

Paradoxical effects of protein synthesis inhibitors on uridine uptake in cultured cells: Possible role of uncharged tRNA in regulating metabolism

Previous studies (J. Biol. Chem, 253: 99–105, 1978) showed that thyrotropin-releasing hormone (TRH) acutely stimulated uridine uptake in pituitary cell (GH₄C₁) cultures. Studies on the role of protein synthesis in this response to TRH led to the finding that an inhibitor of ribosomal translation, cycloheximide, also stimulated uridine uptake acutely. Studies reported here attempt to determine the mechanism of cycloheximide action and whether cycloheximide and hormone stimulation of uridine uptake occurred by similar pathways. The experiments presented indicate that: (1) seven inhibitors of ribosomal translation stimulated uridine uptake; (2) in contrast, inhibition of protein synthesis at tRNA aminoacylation resulted in reduced rates of uridine uptake; (3) inhibition of tRNA aminoacylation blocked cycloheximide but not TRH stimulation of uptake; (4) cycloheximide stimulation of uptake was restricted to amino acid-depleted cultures; (5) amino acid supplementation stimulated uridine uptake with a time-course identical to that of cycloheximide; (6) cycloheximide and amino acid supplementation promoted reacylation of cellular tRNAs in amino acid-depleted cultures; and (7) cycloheximide stimulation of uridine uptake resulted from enhanced nucleoside phosphorylation rather than increased uridine transport. We conclude that cycloheximide and amino acid stimulation of uridine phosphorylation may be mediated through a common pathway involving the extent of amino-acylation of cellular tRNAs. Furthermore, cycloheximide and TRH stimulate uridine phosphorylation by pathways that are distinguishable. It is apparent that not all cellular effects of cycloheximde can be attributed solely to inhibition of the synthesis of proteins.     

Inhibition of metabolic effects of growth hormone by various inhibitors of cyclic nucleotide phosphodiesterase

When isolated diaphragms of hypophysectomized rats were incubated with bovine growth hormone in the presence of the cyclic nucleotide inhibitors theophylline, quinine and papaverine, the stimulatory effects of the hormone on leucine incorporation into protein, α-aminoisobutyric acid and 3-O-methylglucose transport were suppressed or abolished entirely. The degree of suppression of the hormone effects appeared to correlate with the extent of glycogenolysis caused by the drugs. Thoephylline also rapidly reversed the stimulation of protein synthesis and amino acid and sugar transport produced by growth hormone. When protein synthesis and transport were stimulated by preincubation of the diaphragm with growth hormone, the subsequent addition of theophylline to the medium inhibited the hormonal effects on protein synthesis and sugar transport within 15 min and the effect on amino acid transport within 60 min. These results may mean that the rapid in vitro effects of growth hormone on protein synthesis and membrane transport in rat diaphragm muscle are mediated by a reduction in the cellular level of cyclic AMP or some other nucleotide.Attempts to block the action of growth hormone on 3-O-methylglucose transport by preincubation of the diaphragm with high concentrations (10 mM) of cyclic GMP, cyclic UMP, cyclic TMP and cyclic CMP were unsuccessful. Also an effort was made to mimic the action of growth hormone on sugar transport by incubating the diaphragm with high concentrations of imidazole and histamine, agents known to activate cyclic nucleotide phosphodiesterase. Slight stimulatory effects were obtained, but they could not be correlated with any certainty to the actions of imidazole and histamine on phosphodiesterase.Like growth hormone, insulin also stimulates protein synthesis and amino acid and sugar transport in the isolated rat diaphragm. However, the actions of insulin on these processes were not abolished by theophylline, suggesting some basic difference in the mode of action of these two hormones on protein synthesis and membrane transport in muscle.     

Molecular cloning of mannose-binding lectins from Clivia miniata

Screening of a cDNA library constructed from total RNA isolated from young developing ovaries of Clivia miniata Regel with the amaryllis lectin cDNA clone resulted in the isolation of four different isolectin clones which clearly differ from each other in their nucleotide sequences and hence also in their deduced amino acid sequences. Apparently the lectin is translated from an mRNA of ca. 800 nucleotides encoding a precursor polypeptide of 163 amino acids. Northern blot analysis of total RNA isolated from different tissues of Clivia miniata has shown that the lectin is expressed in most plant tissues with very high lectin mRNA concentrations in the ovary and the seed endosperm.     

大花君子兰大小孢子发生与雌雄配子体发育的研究

该研究运用常规石蜡切片技术,对大花君子兰(Clivia miniata Regel)大、小孢子发生及雌、雄配子体发育进程进行解剖学观察分析,以探讨君子兰生殖生物学解剖特征,为君子兰种子发育和育种提供理论依据。结果表明：(1)大花君子兰花药4室,具分泌型绒毡层。(2)小孢子母细胞减数分裂的胞质分裂为连续型,小孢子四分体为左右对称型,成熟花粉为二细胞型。(3)倒生胚珠,双珠被,厚珠心和雌配子体发育为蓼型。(4)记录了雌雄配子体发育的对应关系,发现雄配子体发育趋于同步,雌配子体发育不同步。(5)开花散粉时,雌配子体尚有处于四核、八核胚囊的时期;成熟胚囊阶段,中央细胞的2个极核位于反足细胞端,反足细胞呈退化状态。具承珠盘结构。     

The stimulatory effect of human chorionic gonadotropin on amino acid uptake by amphibian follicles.

Human chorionic gonadotropin (hCG) stimulates the uptake of eight different amino acids and four nucleosides by Xenopus laevis ovarian follicles. This hormone also stimulates amino acid uptake in the follicles of another amphibian, Callyptocephallela caudiverbera. The stimulation of uptake is due to a reduction in the amino acid concentration required for half-maximal uptake velocity and not to an increment in V_max. The effect of hCG does not require protein synthesis but requires physiological conditions of temperature and pH. Incorporation of radioactive exogenous amino acid into proteins is also stimulated by the hormone, but high-resolution electrophoresis shows that there are no drastic qualitative changes in the pattern of proteins synthesized at early times after hCG treatment. The effect of hCG on the uptake of exogenous amino acids does not appear to be required for oocyte maturation because other hormones such as progesterone and testosterone which induce maturation do not increase amino acid uptake. Also the concentration of hCG required for oocyte maturation is significantly lower than that required for an effect on amino acid transport. Inhibitors of oocyte maturation such as theophylline and cycloheximide do not inhibit the action of hCG on amino acid uptake by the amphibian follicles.     

Amino acid requirement for the growth-hormone stimulation of incorporation of precursors into protein and nucleic acids of liver slices

1. Incorporation of [(14)C]leucine into protein in rat liver slices, incubated in vitro, increased as the concentration of unlabelled amino acids in the incubation medium was raised. A plateau of incorporation was reached when the amino acid concentration was 6 times that present in rat plasma. Labelling of RNA by [(3)H]orotic acid was not stimulated by increased amino acid concentration in the incubation medium. 2. When amino acids were absent from the medium, or present at the normal plasma concentrations, no effect of added growth hormone on labelling of protein or RNA by precursor was observed. 3. When amino acids were present in the medium at 6 times the normal plasma concentrations addition of growth hormone stimulated incorporation of the appropriate labelled precursor into protein of liver slices from normal rats by 31%, and into RNA by 22%. A significant effect was seen at a hormone concentration as low as 10ng/ml. 4. Under the same conditions addition of growth hormone also stimulated protein labelling in liver slices from hypophysectomized rats. Tissue from hypophysectomized rats previously treated with growth hormone did not respond to growth hormone in vitro. 5. No effect of the hormone on the rate or extent of uptake of radioactive precursors into acid-soluble pools was found. 6. Cycloheximide completely abolished the hormone-induced increment in labelling of both RNA and protein. 7. It was concluded that, in the presence of an abundant amino acid supply, growth hormone can stimulate the synthesis of protein in rat liver slices by a mechanism that is more sensitive to cycloheximide than is the basal protein synthesis. The stimulation of RNA labelling observed in the presence of growth hormone may be a secondary consequence of the hormonal effect on protein synthesis. 8. The mechanism of action of growth hormone on liver protein synthesis in vitro was concluded to be similar to its mechanism of action in vivo.     

On the action of sulfanilamide

Summary Studying the action of sulfanilamide on bacterial nitrogen metabolism, it was shown that: a. Sulfanilamide does not alter the rate of gelatin-hydrolysis by papain or by the proteinase ofB. pyocyaneum andB. prodigiosum. b. Sulfanilamide does not influence the synthesis of aspartic acid from fumaric acid and ammonium chloride by restingB. coli. c. Addition of single amino acids does not counteract sulfanilamide. d. Addition of single amino acids merely accelerates growth slightly; a marked acceleration was obtained only by adding various amino acids simultaneously. e. The addition of such an optimal mixture of amino acids did not exert any influence on the action of sulfanilamide on growth. As the growth acceleration shows that the bacteria are saved an important output of energy in synthesis as a result of the supply of the amino acids, we conclude that sulfanilamide action cannot be due to interference with the synthesis of amino acids from inorganic nitrogen (f.i. NH₂ + pyruvate).Considering these facts, we expect sulfanilamide to pursuit its action on bacterial growth by interfering with protein anabolism, anywhere in the synthesis of protein from amino acids.     

Hormonal modulation of reproduction-specific thiamin carrier protein in the rat

The hormonal modulation of thiamin carrier protein in the plasma and uterine luminal secretion during the normal reproductive phases of the animal (estrous cycle and pregnancy) as well as during experimental estrogenisation was investigated in the rat using a specific and sensitive homologous radioimmunoassay procedure developed for this purpose. Following a single injection of estrogen to immature male rats, thiamin carrier protein rapidly accumulated in plasma attaining peak concentration at 48 h and declining thereafter. A 1.5-fold amplification of the inductive response was observed on secondary stimulation with the hormone. The magnitude of the response exhibited a clear dependency on the dose of the steroid hormone, whereas the time at which peak levels of thiamin carrier protein production was remained unaltered in the concentration range of the steroid tested. The inductive effect of estrogen was severely curtailed by the antiestrogens,viz., En- and Zu-clomiphene citrates, while progesterone was incapable of either modulating the estrogen-induced response or eliciting an induction by itself. Cycloheximide drastically blocked the response to estrogen. Evidence for the ability of uterus to serve as yet another independent site of thiamin carrier protein synthesis was obtained byin vitro incorporation of radioactive amino acids into immunoprecipitable thiamin carrier protein in the tissue explants of estrogenised female rats. The levels of thiamin carrier protein in uterine luminal fluid measured during estrous cycle, pregnancy and experimental estrogenisation exhibited remarkable similarity to the plasma thiamin carrier protein profiles.     

A self-inhibitor of protein synthesis in the conidia of Glomerella cingulata

Summary A diffusible self-inhibitor of germination of conidia of Glomerella cingulata appears to act as a regulator of protein synthesis. Both uptake of labeled amino acids and their incorporation into protein are reduced by the inhibitor or by crowding. Compared to conidia incubated without self-inhibitor, conidia incubated with self-inhibitor incorporated no labeled amino acids into protein in the first hour and 80% less in 6h. Thoroughly washed conidia were more permeable to amino acids and incorporated 6 times more precursor into proteins than unwashed conidia. At high density in nutrient medium, conidia of G. cingulata preferentially form secondary conidia instead of germ tubes and a mycelium. This inhibition of germination of conidia and regulation of development is mimicked by exposing them to an auto-inhibitor extracted from used culture medium and conidial washings. Germination of conidia of G. cingulata involves two steps, an initial step of 5 h duration which continues unaffected by crowing (1.7×10⁸/ml) and a subsequent 2 h step which conidia do not take unless they are sufficiently diluted. It is this step for which protein synthesis may be required.Non-Standard Abbreviations CHM cyloheximide - NM Neurospora minimal medium - psi pound per square inch - RPH reconstituted algal protein hydrolysate - TCA trichloroacetic acid     

Studies on phosphatidylinositol phosphodiesterase (phospholipase C type) of Bacillus cereus. II. In vivo and immunochemical studies of phosphatase-releasing activity.

Protein synthesis in the cultured rat pineal gland was monitored during the course of N-acetyltransferase induction by (l-isoproterenol or dibutyryl cyclic AMP. The incorporation of labeled amino acids into gland protein was essentially linear over a 6-h experimental period. Examination of the newly synthesized proteins by polyacrylamide gel electrophoresis and autoradiography did not reveal the appearance nor the disappearance of any specific protein(s) caused by (l-isoproterenol or (l)-propranolol. The lack of stimulation of synthesis of any specific protein was further demonstrated by constant ratio of incorporation in double-label experiments. Either 2μm (l)-isoproterenol or 1 mm dibutyryl cyclic AMP stimulated protein synthesis 20–40%. This increase was not due to an enhanced uptake of precursor radiolabeled amino acids by the glands when incubated with the β-agonist or cyclic AMP derivative. The stimulation of protein synthesis caused by (l)-isoproterenol was abolished by the β-antagonist (l)-propranolol. These results suggest that β-agonists may increase pineal gland protein synthesis through their relevant receptor and the generation of cyclic AMP. This increase in synthesis appears to be general and no selective elevation increase in any one band was observed.     

Mutational analysis of the Escherichia coli spoT gene identifies distinct but overlapping regions involved in ppGpp synthesis and degradation

The spoT gene of Escherichia coli encodes a guanosine 3′,5′-bis(diphosphate) 3′-pyrophosphohydrolase (ppGppase) as well as an apparent guanosine 3′,5′-bis(diphosphate) synthetase (designated PSII). To determine the regions of the SpoT protein that are required for these two competing activities, we analysed plasmid-borne deletion mutations for their ability to complement chromosomal mutations defective in each activity. We found that a region containing the first 203 amino acids of the 702-amino-acid SpoT protein was sufficient for ppGppase activity while an overlapping region containing residues 67–374 was sufficient for PSII activity. These data indicate that the catalytic sites involved in the two activities are separate but closely linked in the primary sequence of the SpoT protein. A ppGppase-defective Δ1–58 deletion mutant strain failed to synthesize ppGpp in response to nutrient limitation, also supporting the notion that PSII activity from wild-type SpoT does not increase in response to nutrient limitation. Using a strain lacking PSII activity but retaining ppGppase activity, we determined the contribution of the RelA protein (ppGpp synthetase I, PSI) to ppGpp synthesis following glucose starvation. We found that the RelA protein activity accounts for the initial burst of ppGpp synthesis at the onset of glucose starvation but that this source of synthesis is absent when amino acids are present during glucose starvation.     

PROTEIN BIOSYNTHESIS IN SALT-SHOCKED CELLS OF STICHOCOCCUS BACILLARIS (CHLOROPHYCEAE)1

We have developed an in vivo¹⁴C-amino acid labelling procedure for monitoring protein synthesis in salt-shocked cells of Stichococcus bacillaris Naeg. This alga possesses an efficient transport system for the uptake of leucine, methionine, and phenylalanine and rapidly incorporates these amino acids into proteins. Of the three amino acids tested, ¹⁴C-phenylalanine is ideally suited for labelling proteins in S. bacillaris, as it establishes an early equilibrium between uptake and incorporation of the amino acid into proteins. The uptake of phenylalanine shows little inhibition following transfer of cells to higher salinities and is also not affected in short-term experiments by the presence of the protein inhibitors cycloheximide and chloramphenicol. While Stichococcus bacillaris grows slowly at salinities equal to, or higher than, 150% artificial seawater (ASW), it shows surprising rates of recovery of major physiological functions following considerable salt shocks. Cells transferred from 33 to 150% ASW show complete recovery of photosynthetic activity and protein synthesis within 10–15 min, and cell transferred from 33 to 300% ASW recover 50% of their capacity to synthesize proteins within. 1 h. Cytoplasmic and organellar protein synthesis appears to be equally sensitive to the effects of salt shocks according to studies with protein synthesis inhibitors.     

Albumin synthesis during induced and spontaneous metamorphosis in the bullfrog Rana catesbeiana

Treatment of premetamorphic tadpoles with triiodothyronine (T₃) alters the in vivo distribution of radioactive amino acids among serum protein fractions. The effects on the albumin fraction have been interpreted as reflections of the relative rate of synthesis. About 12 hr after intraperitoneal injection of 2.5 × 10^?10 mole of T₃ per gram, there is an increase in the relative rate of albumin synthesis. The effect peaks on day 3 at 5 × the untreated level and returns to near the untreated level by day 6. Continuous immersion in 1 × 10^?7M T₃ results in a similar stimulation of albumin synthesis, but with no decline after day 3. The timing of the response is independent of dose or route of T₃ administration. The effect of T₃ on the relative rate of albumin synthesis is also observed in froglets. There is a 6-fold increase in the relative rate of albumin synthesis during spontaneous metamorphosis peaking at stage XXI and returning to the premetamorphic level by stage XXV. The following was concluded: (1) The increase in the relative rate of albumin synthesis during metamorphosis results from increased endogenous thyroid levels. (2) Following a peak, the decline in albumin synthesis observed in induced and spontaneously metamorphosing animals is a result of decreasing thyroid hormone levels. (3) The effect of T₃ on albumin synthesis may be the summation of two effects, a direct effect of T₃ and a stimulation by amino acids from the resorbing tail. (4) A decreased relative rate of albumin degradation or a sparing of albumin is probably responsible for the elevated relative concentration of albumin in the serum of postmetamorphic animals.     

Plasma free amino acid kinetics in rainbow trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) using a bolus injection of <Superscript>15</Superscript>N-labeled amino acids

To gain insight into the metabolic design of the amino acid carrier systems in fish, we injected a bolus of ¹⁵N amino acids into the dorsal aorta in mature rainbow trout (Oncorhynchus mykiss). The plasma kinetic parameters including concentration, pool size, rate of disappearance (R _d), half-life and turnover rate were determined for 15 amino acids. When corrected for metabolic rate, the R _d values obtained for trout for most amino acids were largely comparable to human values, with the exception of glutamine (which was lower) and threonine (which was higher). R _d values ranged from 0.9 μmol 100 g⁻¹ h⁻¹ (lysine) to 22.1 μmol 100 g⁻¹ h⁻¹ (threonine) with most values falling between 2 and 6 μmol 100 g⁻¹ h⁻¹. There was a significant correlation between R _d and the molar proportion of amino acids in rainbow trout whole body protein hydrolysate. Other kinetic parameters did not correlate significantly with whole body amino acid composition. This indicates that an important design feature of the plasma-free amino acids system involves proportional delivery of amino acids to tissues for protein synthesis.     

Identification of critical amino acids involved in α1-β interaction in voltage-dependent Ca channels

In voltage-dependent Ca²⁺ channels, the α₁ and β subunits interact via two cytoplasmic regions defined as the Alpha Interaction Domain (AID) and Beta Interaction Domain (BID). Several novel amino acids for that interaction have now been mapped in both domains by point mutations. It was found that three of the nine amino acids in AID and four of the eight BID amino acids tested were essential for the interaction. Whereas the important AID amino acids were clustered around five residues, the important BID residues were more widely distributed within a larger 16 amino acid sequence. The affinity of the AID_A GST fusion protein for the four interacting β_1b BID mutants was not significantly altered compared with the wild-type β_1b despite the close localization of mutated residues to disruptive BID amino acids. Expression of these interactive β mutants with the full-length α_1A subunit only slightly modified the stimulation efficiency when compared with the wild-type β_1b subunit. Our data suggest that non-disruptive BID sequence alterations do not dramatically affect the β subunit-induced current stimulation.