Incorporation Of 3H-phenylalanine Research Articles

Validation of the flooding dose technique to determine fractional rates of protein synthesis in a model bivalve species, the blue mussel (Mytilus edulis L.)

For the first time, use of the flooding dose technique using 3H-Phenylalanine is validated for measuring whole-animal and tissue-specific rates of protein synthesis in the blue mussel Mytilus edulis (61mm shell length; 4.0g fresh body mass). Following injection, the phenylalanine-specific radioactivities in the gill, mantle and whole-animal free pools were elevated within one hour and remained elevated and stable for up to 6h following injection of 3H-phenylalanine into the posterior adductor muscle. Incorporation of 3H-phenylalanine into body protein was linear over time following injection and the non-significant intercepts for the regressions suggested incorporation into body protein occurred rapidly after injection. These results validate the technique for measuring rates of protein synthesis in mussels. There were no differences in the calculated rates following 1–6h incubation in gill, mantle or whole-animal and fractional rates of protein synthesis from the combined time course data were 9.5±0.8%d−1 for the gill, 2.5±0.3%d−1 for the mantle and 2.6±0.3%d−1 for the whole-animal, respectively (mean values±SEM). The whole-animal absolute rate of protein synthesis was calculated as 18.9±0.6mg protein day−1. The use of this technique in measuring one of the major components of maintenance metabolism and growth will provide a valuable and convenient tool in furthering our understanding of the protein metabolism and energetics of this keystone marine invertebrate and its ability to adjust and respond to fluctuations, such as that expected as a result of climate change.

Effect of hydroxyurea on mitotic activity 3H-thymidine and 3H-phenylalanine incorporation in the antheridial filament cells of Chara vulgaris

Hydroxyurea inhibits mitotic activity in cells of the antheridial filaments of <i>Chara vulgaris</i> by blocking phase S and phase G<sub>2</sub>. Blocking of cells in phase G<sub>2</sub> also occurs in the case of the root meristem cells of <i>Helianthus annuus</i> and <i>Vicia faba</i> var. <i>minor</i>. <sup>3</sup>H-thymidine incorporation confirmed autoradiographically the blocking of cells of the antheridial filaments in <i>Chara vulgaris</i> at phase S and slowing down of the rate of DNA replication. Incubation with <sup>3</sup>H-phenylalanine demonstrated that hydroxyurea inhibits protein synthesis.

Resveratrol prevents norepinephrine induced hypertrophy in adult rat cardiomyocytes, by activating NO-AMPK pathway

Increased adrenergic drive is a major factor influencing the development of pathological cardiac hypertrophy, a stage which precedes overt heart failure. We examined the effect of resveratrol, a polyphenol (found predominantly in grapes), in preventing norepinephrine induced hypertrophy of adult cardiomyocyte, and the role of nitric oxide (NO) and adenosine monophosphate kinase (AMPK) in the effects of resveratrol. Cardiomyocytes isolated from adult rats were pretreated, or not, with resveratrol and then exposed to norepinephrine for 24h. In other experiments cardiomyocytes were also treated with different pharmacological inhibitors of NO synthase, AMPK and sirtuin for elucidating the signaling pathways underlying the effect of resveratrol. In order to validate the role of these signaling molecules in the in vivo settings, we also examined hearts from resveratrol treated spontaneously hypertensive rats (SHR), a genetic model of essential hypertension. Cardiomyocyte hypertrophy was determined by morphometry and 3H-phenylalanine incorporation assay. NO levels and AMPK activity were measured using a specific assay kit and western blot analysis respectively. In vitro, resveratrol prevented the norepinephrine-induced increase in cardiomyocytes size and protein synthesis. Pharmacological inhibition of NO-AMPK signaling abolished the anti-hypertrophic action of resveratrol. Consistent with the in vitro findings, the anti-hypertrophic effect of resveratrol in the SHR model was associated with increases in NO and AMPK activity. This study demonstrates that NO-AMPK signaling is linked to the anti-hypertrophic effect of resveratrol in adult cardiomyocytes in vitro, and in the SHR model in vivo.

PDGF-BB protects cardiomyocytes from apoptosis and improves contractile function of engineered heart tissue

Platelet-derived-growth-factor-BB (PDGF-BB) can protect various cell types from apoptotic cell death, and induce hypertrophic growth and proliferation, but little is known about its direct or indirect effects on cardiomyocytes. Cardiac muscle engineering is compromised by a particularly high rate of cardiomyocyte death. Here we hypothesized that PDGF-BB stimulation can (1) protect cardiomyocytes from apoptosis, (2) enhance myocyte content in and (3) consequently optimize contractile performance of engineered heart tissue (EHT). We investigated the effects of PDGF-receptor activation in neonatal rat heart monolayer- and EHT-cultures by isometric contraction experiments, cytomorphometry, 3H-thymidine and 3H-phenylalanine incorporation assays, quantitative PCR (calsequestrin 2, α-cardiac and skeletal actin, atrial natriuretic factor, α- and β-myosin heavy chain), immunoblotting (activated caspase 3, Akt-phosphorylation), and ELISA (cell death detection). PDGF-BB did not induce hypertrophy or proliferation in cardiomyocytes, but enhanced contractile performance of EHT. This effect was concentration-dependent (Emax 10ng/ml) and maximal only after transient PDGF-BB stimulation (culture days 0–7; total culture duration: 12days). Improvement of contractile function was associated with higher cardiomyocyte content, as a consequence of PDGF-BB mediated protection from apoptosis (lower caspase-3 activity particularly in cardiomyocytes in PDGF-BB treated vs. untreated EHTs). We confirmed the anti-apoptotic effect of PDGF-BB in monolayer cultures and observed that PI3-kinase inhibition with LY294002 attenuated PDGF-BB-mediated cardiomyocyte protection. We conclude that PDGF-BB does not induce hypertrophy or proliferation, but confers an anti-apoptotic effect on cardiomyocytes. Our findings suggest a further exploitation of PDGF-BB in cardiomyocyte protection in vivo and in vitro.

Effect of diabetes on the activity of soluble factors required in protein synthesis.

The effect of chronic diabetes on the ability of soluble factors to support ribosomal protein synthesis was studied. It was found that the hepatic supernatant fraction prepared from pancreatectomized diabetic rats was less efficient than normal in catalyzing the incorporation of 3H-phenylalanine into microsomal protein. The ability to incorporate radioactivity from aminoacyl-sRNA into ribosomal protein, however, was unchanged in diabetes. Subsequent experiments confirmed the impression that the formation of aminoacyl-sRNA is depressed in the hepatic 30,000 rpm supernatant prepared from diabetic animals.

Luminal Threonine Concentration Acutely Affects Intestinal Mucosal Protein and Mucin Synthesis in Piglets 3

Intestinal mucin synthesis is sensitive to dietary threonine supply, which suggests that the gut’s requirement for threonine may comprise a significant proportion of the whole body requirement. We used a continuously perfused gut loop model and intraluminal flooding dose technique in 6 young pigs to study the acute effects of varying luminal availability of threonine on intestinal protein and mucin syntheses. A complete amino acid mixture containing 0, 21, or 56 mg threonine/g total amino acids (whole body requirement ratio is ∼28 mg/g) was continuously perfused in isolated loops for 120 min, including a 30-min 3H-phenylalanine flooding dose. We measured fractional synthesis rates of total mucosal protein and mucin by analyzing 3H-phenylalanine incorporation. Fractional rates of total mucosal protein synthesis were higher in loops perfused with solutions containing threonine at 56 mg/g (66 ± 4%/d) compared with 0 mg/g (42 ± 9%/d) and 21 mg/g (53 ± 6%/d) (P < 0.05). For mucin, fractional rates of synthesis differed between 0 mg/g (323 ± 72%/d), 21 mg/g (347 ± 49%/d), and 56 mg/g (414 ± 31%/d) (P < 0.05). In addition, total proline and threonine concentrations in the protein hydrolysates increased with luminal threonine concentration (P < 0.05), indicating an increase in threonine- and proline-rich proteins. De novo synthesis of mucosal and mucin proteins is acutely sensitive to luminal threonine concentration, which demonstrates the importance of dietary amino acid supply to gut protein metabolism.

Endothelin-1 induced hypertrophic effect in neonatal rat cardiomyocytes: Involvement of Na +/H + and Na +/Ca 2+ exchangers

Endothelin-1 (ET-1) is a potent agonist of cell growth that also stimulates Na +/H + exchanger isoform 1 (NHE-1) activity. It was hypothesized that the increase in intracellular Na + ([Na +] i) mediated by NHE-1 activity may induce the reverse mode of Na +/Ca 2+ exchanger (NCX rev) increasing intracellular Ca 2+ ([Ca 2+] i) which in turn will induce hypertrophy. The objective of this work was to test whether the inhibition of NHE-1 or NCX rev prevents ET-1 induced hypertrophy in neonatal rat cardiomyocytes (NRVMs). NRVMs were cultured (24 h) in the absence (control) and presence of 5 nmol/L ET-1 alone, or combined with 1 μmol/L HOE 642 or 5 μmol/L KB-R7943. Cell surface area, 3H-phenylalanine incorporation and atrial natriuretic factor (ANF) mRNA expression were increased to 131 ± 3, 220 ± 12 and 190 ± 25% of control, respectively ( P < 0.05) by ET-1. [Na +] i and total [Ca 2+] i were higher (8.1 ± 1.2 mmol/L and 636 ± 117 nmol/L, respectively) in ET-1-treated than in control NRVMs (4.2 ± 1.3 and 346 ± 85, respectively, P < 0.05), effects that were cancelled by NHE-1 inhibition with HOE 642. The rise in [Ca 2+] i induced by extracellular Na + removal (NCX rev) was higher in ET-1-treated than in control NRVMs and the effect was prevented by co-treatment with HOE 642 or KB-R7943 (NCX rev inhibitor). The ET-1-induced increase in cell area, ANF mRNA expression and 3H-phenylalanine incorporation in ET-1-treated NRVM were decreased by NHE-1 or NCX rev inhibition. Our results provide the first evidence that NCX rev is, secondarily to NHE-1 activation, involved in ET-1-induced hypertrophy in NRVMs .

P0585 ROTAVIRUS ENTERITIS OF MALNOURISHED PIGS INCREASES INTESTINAL ACTIVITY OF RIBOSOMAL P70 S6 KINASE, INCREASES GUT PROTEIN SYNTHESIS AND REDUCES MUSCLE PROTEIN SYNTHESIS

Introduction: The rate of protein synthesis in eukaryotic cells is controlled by the mammalian target of rapamycin (mTOR/FRAP). mTOR regulates the activity of ribosomal protein s6 kinase (p70s6k) and ribosomal protein 4E-BP1. mTOR activity has been called the “amino acid sensor” of eukaryotic cells. Because intestinal cell migration after wounding is markedly reduced by cycloheximide and rapamycin, we postulated that protracted diarrhea of infancy in malnourished hosts is mediated by downregulation of mTOR/p70s6k. Methods: Eight non-infected control pigs received full-strength formula. Eight infected pigs received a 50% restriction of formula, which we have previously shown results in “protracted diarrhea of infancy.” Infection was initiated by inoculation with porcine rotavirus on day 6 of life. We compared rates of ex vivo ileal and semitendonosus muscle 3H-phenylalanine incorporation into protein, and we quantified phosphorylation of p70s6k (by western blot)(probed with antibody to phospho-Thr421/Ser424) on days 0, 1, 3, 5,and 11 post-infection (p.i.). Results: Because feeding markedly activated p70s6k in pig ileum (n=3 additional pigs), all subjects were studied after a 9-h fast. At each time-point p.i., intestinal protein synthetic rate in infected pigs was increased 30–50%, while muscle protein synthesis decreased 30–50% compared with control pigs. Peaking on days 3 and 5 post-infection, the activation level of p70s6k was increased >4-fold in intestinal tissue. The p85 isoform (nuclear p70s6k) was activated significantly more than the p70 isoform (cytoplasmic) by infection, with a peak activation on day 3. Conclusion: Our results support dynamic control of intestinal protein synthesis and p70s6k by feeding. Furthermore, infectious enteritis results in increased bowel protein synthesis, in parallel to significant activation of p70s6k, while muscle protein synthesis is coordinately reduced. The increase in intestinal protein synthesis and p70s6k activity occurs in an epithelium enriched with metabolically active, proliferating crypt cells.

Redox regulation of MAPK pathways and cardiac hypertrophy in adult rat cardiac myocyte

OBJECTIVESWe analyzed the regulatory function of reactive oxygen species (ROS) on the hypertrophic signaling in adult rat cardiac myocytes.BACKGROUNDThe ROS regulate mitogenic signal transduction in various cell types. In neonatal rat cardiac myocyte, antioxidants have been shown to inhibit cardiac hypertrophy, and ROS are suggested to modulate the hypertrophic signaling. However, the conclusion may not reflect the situation of mature heart, because of the different natures between neonatal and adult cardiac myocytes.METHODSCultured adult rat cardiac myocytes were stimulated with endothelin-1 (ET-1) or phenylephrine (PE), and intracellular ROS levels, the activities of mitogen-activated protein kinases (MAPKs; ERK, p38, and JNK), and 3H-phenylalanine incorporation were examined. We also examined the effects of antioxidant pretreatment of myocytes on MAPK activities and cardiac hypertrophy to analyze the modulatory function of redox state on MAPK-mediated hypertrophic signaling.RESULTSThe ROS levels in ET-1- or PE-stimulated myocytes were maximally increased at 5 min after stimulation. The origin of ROS appears to be from NADH/NADPH oxidase, because the increase in ROS was suppressed by pretreatment of myocytes with NADH/NADPH oxidase inhibitor diphenyleneiodonium. Extracellular signal-regulated kinase (ERK) activity was increased by the stimulation of ET-1 or PE. In contrast, p38 and c-Jun-N-terminal protein kinase (JNK) activities did not change after these stimulations. Antioxidant treatment of myocytes suppressed the increase in ROS and blocked ERK activation and the subsequent cardiac hypertrophy induced by these stimuli.CONCLUSIONSThese data demonstrate that ROS mediate signal transduction of cardiac hypertrophy induced by ET-1 or PE in adult rat cardiac myocytes.

Transgenic Hypersecretion of des(1–3) Human Insulin-Like Growth Factor I in Mouse Milk Has Limited Effects on the Gastrointestinal Tract in Suckling Pups

We tested the hypothesis that chronic ingestion of increased concentrations of milk-borne des(1–3) human insulin-like growth factor-I (hIGF-I) stimulates gastrointestinal growth and development in suckling mice. We used a transgenic mouse with targeted, lactation-dependent, overexpression of des(1–3) hIGF-I in the mammary gland (IGF). Pups were suckled (7 pups per litter) from birth by either IGF (n = 3–6 litters) or control (n = 3–5 litters) dams. In IGF and control pups, we measured the growth (protein and DNA content) and protein synthesis rate (3H-phenylalanine incorporation) of gastrointestinal and visceral organs in 4-, 8-, 12-, 16- and 29-d-old pups. Des(1–3) hIGF-I in milk from IGF dams was 40–200-fold higher than mouse IGF in either IGF or control dams, but was not detected in the plasma of pups suckling IGF dams. Small intestinal weight, protein and DNA content at 8 and 16 d were greater in pups suckling IGF dams than control dams; protein synthesis was also greater in IGF pups at 8 d. Total intestinal lactase activity at 8 and 12 d of age tended to be higher (P < 0.10) in IGF than in control pups. Hypersecretion of des(1–3) hIGF-I in milk ingested by suckling mice pups had limited effects on the growth and maturation of the gastrointestinal tract. Moreover, there was little evidence that milk-borne IGF-I is absorbed into the circulation and stimulates visceral organ growth. This study also demonstrates the feasibility of using mammary-specific transgenes to increase the concentration of milk-borne growth factors to examine whether they affect the growth and development of the suckling neonate.

Dietary Fiber and Short-Chain Fatty Acids Affect Cell Proliferation and Protein Synthesis in Isolated Rat Colonocytes

Colonic metabolism may be affected by dietary fiber and short-chain fatty acids, the products of fiber fermentation. The aim of this study was to assess the effects of fiber supplementation (150 g/kg diet) on dynamic measurements of metabolism in isolated rat colonic epithelial cells. Additionally, we investigated the effect of in vitro short-chain fatty acid and glutamine concentrations and media osmolarity on oxygen uptake, protein synthesis, cell proliferation and anaplerotic flux. Colonocyte oxygen consumption did not differ due to fiber supplementation or the inclusion of short-chain fatty acids in incubation media. Cell proliferation (3H-thymidine uptake) was increased by fiber consumption (P ≤ 0.05) but was decreased by supplementing short-chain fatty acids in vitro (P < 0.05), independent of osmolarity. Protein synthesis (3H-phenylalanine incorporation) was unaffected by fiber supplementation but was decreased when short-chain fatty acids were present in incubation media (P ≤ 0.05). However, this decrease was also induced by in vitro supplementation with isoosmotic NaCl concentrations. These data show that although fiber supplementation increases in vitro colonocyte proliferation, the unchanged oxygen uptake rate indicates that there was no concurrent increase in energy expenditure.

Sepsis increases production of total secreted proteins, vasoactive intestinal peptide, and peptide YY in isolated rat enterocytes

The effect of sepsis on the synthesis of endogenous and secretory proteins, including vasoactive intestinal peptide (VIP) and peptide YY (PYY), was determined in enterocytes from jejunum of rats. Sepsis was induced by cecal ligation and puncture (CLP). Control rats were sham-operated. Total endogenous and secreted protein synthesis was assessed in incubated jejunal enterocytes by measuring incorporation of 3H-phenylalanine into protein. Release of VIP and PYY into the medium of incubated enterocytes and cellular levels of the gut peptides were measured by radioimmunoassay. Sixteen hours after CLP, synthesis rates of both endogenous and secreted proteins were increased, and this effect of sepsis was most pronounced in cells from the lower parts of the villi and crypts. Enterocytes from septic rats released more VIP and PYY into the incubation medium, and approximately half of the peptides they released were newly synthesized VIP and PYY. Intracellular levels of VIP and PYY were increased as early as 4 hours after induction of sepsis. Our results suggest that sepsis stimulates the synthesis of endogenous and secretory proteins, including certain gut peptides, in small intestine mucosa. This is consistent with previous observations of increased circulating levels of VIP, PYY and other gastrointestinal hormones during sepsis. The biological significance of increased synthesis of gut peptides and other intestinal proteins during sepsis remains to be determined.

Effect of glutamine on protein synthesis in isolated intestinal epithelial cells.

The influence of glutamine on protein synthesis in small-bowel enterocytes was tested. Enterocytes were isolated from different levels of the villi of rat jejunum and were incubated in the presence of different glutamine concentrations, up to 3.4 mmol/L. Protein synthesis was determined by measuring incorporation of 3H-phenylalanine into trichloroacetic acid-precipitated proteins. Glutamine, but no other amino acids, stimulated protein synthesis in enterocytes from all levels of the villi. A maximal effect was noted at a glutamine concentration of 0.67 mmol/L, which is the normal plasma concentration. The amino acid stimulated the synthesis of both secreted and nonsecreted proteins. The stimulatory effect of glutamine on protein synthesis was blocked by the glutaminase inhibitor 6-diazo-5-oxo-L-norleucine and was duplicated by equimolar concentrations of acetoacetate or 3-hydroxybutyrate. The results suggest that glutamine stimulates protein synthesis in small-bowel enterocytes and that this effect of glutamine is related to provision of energy. The findings are important because they suggest that increased protein synthesis may be one of the mechanisms by which glutamine exerts its protective effect on gut mucosa during critical illness.

Is the metabolic response to sepsis in skeletal muscle different in infants and adults? An experimental study in rats

In this study we compared the effect of sepsis on muscle protein metabolism in infant (3 to 4 weeks) and adult (3 to 4 months) rats. Sepsis was induced by cecal ligation and puncture (CLP). Control animals underwent sham operation. Sixteen hours after CLP or sham operation, metabolic studies were performed in incubated intact extensor digitorum longus muscles from infant rats or in strips of the same muscle from adult rats. Protein synthesis rate was determined as incorporation of 3H-phenylalanine into protein; total and myofibrillar protein breakdown rates were determined as release of tyrosine and 3-methylhistidine, respectively. Mortality rate following CLP was similar in both age groups. Basal protein synthesis rate was 3 times higher, total protein breakdown rate was 50% higher, and myofibrillar protein breakdown rate was 3 times higher in infant than in adult animals. However, the relative changes in protein turnover rates induced by sepsis were similar in infant and adult rats: protein synthesis rate decreased by approximately 30%, total protein breakdown increased by 40% to 50%, and myofibrillar protein breakdown increased several-fold. The data suggest that despite prominent differences in basal protein turnover rates between infant and adult rats, the effect of sepsis on muscle protein metabolism is not age dependent.

Inhibition of endothelin-1 induced myocardial protein synthesis by an antisense oligonucleotide against the early growth response gene-1

We explored the role of the recently discovered "early growth response gene-1 (Egr-1)" in the induction of myocardial protein synthesis by endothelin-1. Endothelin-1 stimulated protein synthesis (i.e. 3H-phenylalanine incorporation) in isolated adult rat cardiomyocytes more than 2-fold. Addition of a 15mer Egr-1 antisense oligodeoxyribonucleotide complementary to the first 5 codons of the Egr-1 mRNA completely blocked endothelin-induced protein synthesis. A single base mismatch in the oligonucleotide sequence abolished the inhibitory effect. T3-induced stimulation of protein synthesis was unaffected by the antisense oligonucleotide. These results indicate that the Egr-1 gene product is involved (putatively as a third messenger) in the signal transduction cascade initiated by endothelin-1 which eventually culminates in the induction of cardiac protein synthesis.

Effects of reduced renal function and dietary protein on muscle protein synthesis

We describe a rat model of renal failure that separates catabolic and anabolic states from each other. Muscle protein synthesis was compared during the anabolic period between sham (S) operated and renal failure (RF) rats that were fed different levels of dietary protein. Male rats weighing between 60 and 80 g first had a partial left nephrectomy and then were given a tryptophan deficient diet from four to six days to induce weight loss. On the second day of the diet either a renal decapsulation (S rats) or a simple right nephrectomy (RF rats) was done to enhance the catabolic response in both and to induce renal failure in the RF rats. Following the period of feeding the deficient diet, both groups were fed a nutritionally complete 14, 17, 20 or 30% protein diet for three to five days. This induced a brisk anabolic response as measured by weight gain. Differences in body weight between the S and RF rats after three to five days on the repletion diet generally was less than 10%. The rats then were fasted overnight, fed a standard meal and muscle protein synthesis (Sm%) was measured two hours post-feeding. Sm% was estimated from the incorporation of 3H phenylalanine (PHE) into muscle 10 minutes following the i.v. injection of 3HPHE (25 muCi/100 g body wt) with carrier PHE to flood all the precursor amino acid pools. Weight loss in the catabolic phase was greater and the net weight gain for the two phases was less in the RF group. Overall, renal failure resulted in a significant reduction in Sm% (P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Muscle protein turnover in chickens selected for increased growth rate, food consumption or efficiency of food utilisation: Effects of genotype and relationship to plasma IGF‐I and growth hormone

1. Rates of muscle protein turnover, growth, and food consumption were determined in 4 lines of chickens selected for either weight gain (line W), food consumption (line F), efficiency of food conversion (line E), or at random (line C) and in two Australian commercial broiler strains (S and H). These measures were related to body composition and the circulating concentrations of plasma growth hormone (GH) and IGF-I. 2. N tau-methylhistidine excretion was 10-14% higher in line F and 7-13% lower in line E compared to line C, showing divergence in the rate of muscle protein breakdown with selection. 3. There were no differences between the 4 experimental lines (W, F, E and C) in muscle protein fractional synthesis rates, whether calculated from N tau-methylhistidine excretion or measured directly by 3H-phenylalanine incorporation. 4. No consistent differences were found between lines in circulating concentrations of either GH or IGF-I but plasma IGF-I concentrations were positively correlated over all lines with protein accretion rates. There was a strong inverse correlation over all lines between the rates of protein degradation and FCR. 5. The correlated responses in protein degradation rates are consistent with the notion of a positive genetic association between the overall efficiency of food utilisation for growth and the efficiency of protein metabolism.

Immediate decrease in apparent translation rate caused by salinity change in scyphistomae of aurelia aurita (linnaeus)

1. Abrupt decrease or increase of salinity is followed by an immediate drop in incorporation of 3H phenylalanine into the TCA-insoluble protein fraction, but no major change in oxygen consumption rate. 2. Decrease in salinity at constant osmolarity is not followed by lower incorporation. 3. Recovery from osmotic swelling after salinity decrease coincides with reestablishment of a new stable rate of incorporation, which is only 50–70% of the original rate. 4. Inhibition of translation with continued proteolysis could provide free amino acids for cell volume control.

Autoradiographic comparison of neuronal and glial protein metabolism in rat hippocampus after food-motivated or footscock-motivated conditioning

Quantitative autoradiography has shown that initial food-motivated conditioning results in an increase in 3H-phenylalanine incorporation into cytoplasmic proteins of rat hippocampal neurons. After 3 daily conditioning trials, the incorporation returned to an active control (pseudoconditioning) level while after 6 daily trials, the incorporation was decreased. No changes were revealed in the cells of hippocampal perineuronal glia. Four hours after a footshock-motivated passive avoidance trial, incorporation of 3H-phenylalanine was increased both in the neurons and in their perineuronal glia of rat hippocampus. By the time of a consolidation of this conditioning, such increase still remained in the glia but disappeared in the neurons. Subsequently, no changes were found in the hippocampal neurons or in their perineuronal glia. An importance of the emotional background is outlined for a participation of glial cells in learning-induced metabolic changes in the nervous system.

Phenylalanine hydroxylase deficiency in the peripheral lymphocytes from phenylketonuria.

A radiograph of the incorporation of 3H-phenylalanine and 3H-tyrosine into cultured lymphocytes in a phenylketonuria patient indicates phenylalanine hydroxylase deficiency in the peripheral lymphocytes. Cultured lymphocytes from the patient showed much less uptake of 3H-phenylalanine and a slightly higher incorporation of 3H-tyrosine than the control. It is possible that a lack of phenylalanine hydroxylase in the peripheral lymphocytes of PKU patients prevents oxidation of phenylalanine to tyrosine.